BR122019023058B1 - METHOD FOR INCREASING THE EFFICIENCY IN THE USE OF NITROGEN, PRODUCTION, BIOMASS, GROWTH RATE, VIGOR, OIL CONTENT, AND / OR TOLERANCE TO THE ABIOTIC STRESS OF AN ISOLATED NUCLEIC ACID PLANT AND CONSTRUCTION - Google Patents

Abstract

method for increasing efficiency in the use of nitrogen, production, biomass, growth rate, vigor, oil content, and / or tolerance to abiotic stress of a plant, isolated nucleic acid construct, isolated polypeptide, method for growing a crop , provides methods to increase efficiency in the use of nitrogen, production, biomass, growth rate, vigor, oil content, fiber production, fiber quality and / or tolerance to abiotic stress of a plant expressing an exogenous polynucleotide in the plant, comprising a nucleic acid sequence at least 80% homologous to sequence id: 387, 1-386, 388-469, 763-3704 and 3705; or an exogenous polynucleotide that encodes a polypeptide at least 80% identical to sequence number: 602, 470-601, 603-762, 3706-5858, 5860-5910, 5912, 5914-5923, 5925-6046 or 6047; an isolated polynucleotide is also provided, comprising a nucleic acid sequence selected from a group consisting of sequence number 387, 1-386, 388-469, 763-3704 and 3705, which can be used to increase efficiency in the use of nitrogen, production, biomass, growth rate, vigor, oil content, fiber production, fiber quality and / or tolerance to abiotic stress in a plant.

Description

METHOD FOR INCREASING THE EFFICIENCY IN THE USE OF NITROGEN, PRODUCTION, BIOMASS, GROWTH RATE, VIGOR, OIL CONTENT, AND / OR TOLERANCE TO THE ABIOTIC STRESS OF AN ISOLATED NUCLEIC ACID PLANT AND CONSTRUCTION Divided from BR112014012871-5, deposited on 11/27/2012 FIELD OF APPLICATION AND STATUS OF TECHNIQUE

[001] The present patent application, in some respective applications, refers to the new polynucleotides and polypeptides that can increase efficiency in the use of nitrogen, efficiency in the use of fertilizer, production (for example, seed / grain production , oil production), growth rate, vigor, biomass, oil content, fiber production, fiber quality and / or length, resistance to abiotic stress and / or water use efficiency of a plant.

[002] A common approach to promote plant growth was, and remains, the use of natural nutrients, as well as synthetic nutrients (fertilizers). In this way, fertilizers are the fuel behind the "green revolution", directly responsible for the exceptional increase in crop yields in the last 40 years and considered as the number one general expense of agriculture. Of the three macronutrients supplied as main fertilizers [Nitrogen (N), Phosphate (P) and Potassium (K)], nitrogen often acts as a rate-limiting element in plant growth and all crop areas have a fundamental dependency on inorganic fertilizers nitrogen based. Nitrogen normally needs to be replenished every year, particularly for cereals, which comprise more than half of the cultivated areas worldwide. For example, nitrogen-based inorganic fertilizers, such as ammonium nitrate, potassium nitrate or urea, typically account for 40% of the costs associated with crops, such as corn and wheat.

[003] Nitrogen is an essential macronutrient for the plant, responsible for the biosynthesis of nucleic acids and amino acids, prosthetic groups, plant hormones, chemical defenses of the plant, etc. In addition, nitrogen often acts as a limiting factor in the growth rate of the plant and all field crops have a fundamental dependence on inorganic nitrogen. In this way, nitrogen is translocated to the bud, where it is stored in the leaves and stem during the rapid stage of plant development, and therefore until it blooms. In corn, for example, plants accumulate the volume of their organic nitrogen during the germination period of the grain and until it blooms. Once the fertilization of the plant occurs, the grains begin to form and become the main receptacle for the plant's nitrogen. The stored nitrogen can then be redistributed from the leaves and stem that served as storage compartments until grain formation.

[004] As fertilizer runs out quickly in most soil types, it must be supplied to growing crops two or three times during the growing season. In addition, the low efficiency in the use of nitrogen (NUElnitrogen use efficiency) of the main crops (for example, in the range of only 30-70%) negatively affects the investment costs of the farmer due to the excess of fertilizer applied. In addition, excessive and inefficient uses of fertilizers are the main factors responsible for environmental problems, such as eutrophication of groundwater, lakes, rivers and seas and nitrate pollution in drinking water, which can cause methemoglobinemia, phosphate pollution, air pollution and the like. However, despite the negative impact of fertilizers on the environment and the limits on fertilizer use that have been controlled by law in several countries, the use of fertilizers is expected to increase in order to support food and fiber production due to rapid population growth and limited land resources. For example, it is estimated that by 2050, more than 150 million tons of nitrogen-based fertilizers will be used worldwide each year.

[005] Increased efficiency in the use of nitrogen by plants should enable the cultivation of crops with a lower investment in fertilizers, or, alternatively, cultivation in lower quality soils and thus have a significant economic impact on both systems agriculture and developing systems.

[006] The generic improvement in the efficiency in the use of fertilizers (FUE 1 fertilizer use efficiency) in plants can be generated either through traditional reproduction or by genetic engineering.

[007] Generation attempts
of plants with improved FUE have been described in American Patent Application No. 20020046419, by Choo, et al .; American Patent Application No. 2005010879, by Edgerton et al .; American Patent Application No. 20060179511, by Chomet et al .; Good, A, et al. 2007 (Engineering nitrogen use efficiency with alanine aminotransferase. Canadian Journal of Botany 85: 252-262); and Good AG et al. 2004 (Trends Plant Sci. 9: 597-605).

[008] Yanagisawa et al. (Proc. Natl. Acad. Sci. U.S.A. 2004 101: 7833-8) describes Dofl transgenic plants that show improved growth under low nitrogen conditions.

[009] American Patent No. 6,084,153, by Good et al. discloses the use of a stress-sensitive promoter to control the expression of Alanine Aminotransferase (AlaAT 1 Alanine Amine Transferase) and transgenic canola plants with improved drought resistance and nitrogen deficiency when compared to control plants.

[0010] The continuous growth of the world population and the deficit of availability of arable land for agriculture affect the yield of plants and products related to plants. Global water supply shortages, desertification, abiotic stress conditions (ABSlabiotic stress) (eg, salinity, drought, flood, underutilized temperature and toxic chemical pollution) and / or limited nitrogen and fertilizer sources cause damage substantial changes to agricultural plantations, such as major changes in plant metabolism, cell death and decreased plant growth and crop productivity.

[0011] Drought is a gradual phenomenon that involves periods of abnormally dry weather that last long enough to produce serious hydrological imbalances, such as crop damage, decreased water supply and increased susceptibility to various diseases.

[0012] Salinity, high levels of salt, affects one in five hectares of irrigated land. None of the five main food crops, eg wheat, corn, rice, potatoes and soybeans, can tolerate excess salt. The harmful effects of salt on plants result both from water deficiency, which leads to osmotic stress (similar to the stress caused by drought), and from the effect of excess sodium ions on important biochemical processes. Like freezing and drought, high amounts of salt cause a water deficit; and the presence of high levels of salt makes it difficult for plant roots to extract water from their environment. Thus, the salination of soils that are used for agricultural production is a significant and growing problem in regions that depend mainly on agriculture and is worsened by excessive use and fertilization and the lack of water, typically caused by climate change and the demands of growing population.

[0013] Suboptimal temperatures affect plant growth and development throughout its life cycle. In this way, low temperatures reduce the germination rate and high temperatures result in leaf necrosis. In addition, mature plants exposed to excess heat can experience heat shock, which can occur in various organs, including leaves and, especially, fruits, when sweating is insufficient to overcome the heat stress. Heat also damages cellular structures, including organelles and the cytoskeleton and impairs membrane function. Heat shock can produce a decrease in the general synthesis of proteins, accompanied by the expression of heat shock proteins, eg, chaperones, which are involved in rearranging heat-denatured proteins. High temperature damage to pollen almost always occurs in conjunction with drought stress and rarely occurs under good irrigation conditions. Combined stress can alter the plant's metabolism in new ways. Excessive cold conditions, eg low temperatures, but above the freezing point, affect crops of tropical origin, such as soy, rice, corn and cotton. Typical cold damage includes wilting, necrotic, chlorine, or loss of ions from cell membranes. Excessive light conditions, which occur under clear atmospheric conditions subsequent to the cold late summer / autumn nights, can lead to photoinhibition of photosynthesis (interruption of photosynthesis). In addition, the cold can lead to loss of production and poor product quality through late ripening of corn.

[0014] The deficiency of nutrients causes adaptations of the root architecture, particularly notable, for example, is the proliferation of the root within areas rich in nutrients to increase the absorption of nutrients. The deficiency of nutrients also causes the activation of metabolic pathways in the plant that maximize the processes of absorption, assimilation and distribution as well as the activation of changes in architecture. Engineering the expression of the triggered genes can cause the plant to show changes in its architecture and improved metabolism, too, under other conditions.

[0015] Furthermore, it is widely known that plants generally respond to water deficiency by creating a system of deeper roots that allow access to moisture located in deeper layers of the soil. The triggering of this effect will allow plants to access nutrients and water located in deeper horizons of the soil, particularly those readily dissolved in water, such as nitrates.

[0016] Production is affected by several factors, such as the number and size of the plant's organs, the architecture of the plant (eg, the number of branches), the established length of the grains, the number of full grains, vigor (eg, seedling), growth rate, root development, use of water, nutrients (eg, nitrogen) and fertilizers and stress tolerance.

[0017] Crops such as corn, rice, wheat, canola and soy account for more than half of the total human caloric intake, either through direct consumption of seeds or through the consumption of animal meat products raised with processed seeds or forage . Seeds are also a source of sugars, proteins, oils and metabolites used in industrial processes. The ability to increase plant production, either by increasing the dry matter accumulation rate, modifying cellulose or lignin composition, increasing stem resistance, increasing meristem size, changing the plant's branching pattern, leaf firmness, increased fertilization efficiency, increased seed dry matter accumulation rate, modified seed development, improved seed filling, or increased oil, starch or protein content in the seeds would have many applications in use agricultural and non-agricultural as well as in the biotechnological production of pharmaceutical products, antibodies or vaccines.

[0018] Studies have shown that adaptations in the plant to adverse environmental conditions are complex genetic traits of a polygenic nature. Conventional culture media and horticultural improvements use selective breeding techniques to identify plants that have desirable characteristics. However, selective breeding is tedious, time consuming and has unpredictable results. In addition, limited germplasmic resources to improve production and incompatibility in crossings between distantly related plant species represent significant problems encountered in conventional breeding. Advances in genetic engineering have enabled man to modify plant germplasm by expressing genes of interest in plants. This technology has the ability to generate crops or plants with improved economic, agronomic or horticultural traits.

[0019] The publication WO N ° 2004/104162 discloses methods to increase the tolerance to abiotic stress and / or biomass in plants generated in this way.

[0020] Publication WO No. 2004/111183 discloses nucleotide sequences to regulate gene expression in trichomes and plant structures and methods using them.

[0021] The publication WO N ° 2004/081173 discloses new sequences and regulatory structures derived from the plant and methods of using such sequences to direct the expression of exogenous polynucleotide sequences in plants.

[0022] The publication WO N ° 2005/121364 discloses polynucleotides and polypeptides involved in the development of plant fibers and the method of using them, in order to increase the quality of the fibers, the production and / or biomass of a fiber producing plant.

[0023] WO publication 2007/049275 discloses isolated polypeptides, polynucleotides that encode them, transgenic plants that express the same and method of using them to increase the efficiency in the use of fertilizers, the tolerance to plant abiotic stress and biomass.

[0024] Publication WO No. 2007/020638 discloses methods to increase tolerance to abiotic stress and / or biomass in plants and plants generated in this way.

[0025] The publication WO No. 2008/122980 reveals structures of genes and methods to increase the oil content, growth rate and biomass of plants.

[0026] WO publication 2008/075364 discloses polynucleotides involved in the development of plant fiber and methods of using them.

[0027] The publication WO N ° 2009/083958 reveals methods to increase the efficiency in the use of water, efficiency in the use of fertilizers, tolerance to biotic / abiotic stress, production and biomass in plants and in plants generated in this way.

[0028] Publication WO No. 2009/141824 discloses isolated polynucleotides and methods for using them to increase the usefulness of the plant.

[0029] Publication WO No. 2009/013750 discloses genes, structures and methods to increase tolerance to abiotic stress, biomass and / or production in plants generated in this way.

[0030] The publication WO N ° 2010/020941 reveals methods to increase the efficiency in the use of nitrogen, tolerance to abiotic stress, production and biomass in plants and in plants generated in this way.

[0031] WO No. 2010/076756 discloses isolated polynucleotides to increase tolerance to abiotic stress, production, biomass, growth rate, vigor, oil content, fiber production, fiber quality and / or efficiency in the use of nitrogen from a plant.

[0032] Publication W02010 / 100595 discloses isolated polynucleotides and polypeptides and methods of using them to increase plant production and / or agricultural characteristics.

[0033] Publication WO No. 2010/049897 discloses isolated polynucleotides and polypeptides and methods of using them to increase plant production, biomass, growth rate, vigor, oil content, tolerance to plant abiotic stress and efficiency in nitrogen use.

[0034] Publication W02010 / 143138 discloses isolated polynucleotides and polypeptides and methods of using them to increase efficiency in the use of nitrogen, efficiency in the use of fertilizers, production, growth rate, vigor, biomass, oil content, tolerance to abiotic stress and / or water use efficiency.

[0035] WO publication No. 2011/080674 discloses isolated polynucleotides and polypeptides and methods of using them to increase plant production, biomass, growth rate, vigor, oil content, tolerance to plant abiotic stress and efficiency in nitrogen use.

[0036] Publication W02011 / 015985 discloses polynucleotides and polypeptides to increase the desirable qualities of the plant.

[0037] Publication W02011 / 135527 discloses isolated polynucleotides and polypeptides and methods of using them to increase plant production and / or agricultural characteristics.

SUMMARY

[0038] According to an aspect of some applications of the present application, a method is provided to increase efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence that encodes a polypeptide, at least 80% identical to SEQ ID. N ° [Sequence Identification N °] 470-762, 3706-5858, 58605910, 5912, 5914-5923, 5925-6046 or 6047, thereby increasing the efficiency in the use of fertilizer (eg, efficiency in the nitrogen use), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to plant abiotic stress.

[0039] In accordance with an aspect of some applications of the present application for a patent, a method is provided to increase the efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence that encodes a polypeptide selected from the group consisting of ID SEQ. N ° 470-762, 3706- 6046 and 6047, thus increasing efficiency in the use of fertilizer (eg efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to plant abiotic stress.

[0040] According to one aspect of some applications of the present application, a method is provided to increase efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence at least 80% identical to the SEQ ID . No. 1-469, 763-3704 or 3705, thereby increasing efficiency in the use of fertilizer (eg efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to plant abiotic stress.

[0041] According to an aspect of some applications of the present application, a method is provided to increase efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress of a plant, comprising expressing within the plant an exogenous polynucleotide comprising the nucleic acid sequence selected from the group consisting of the SEQ ID. N ° 1-469, 763-3704 and 3705, thereby increasing efficiency in the use of fertilizer (eg efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to plant abiotic stress.

[0042] According to an aspect of some applications of the present application, an isolated polynucleotide is provided comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence at least 80% homologous to the sequence of amino acid set out in SEQ ID. 470-762, 3706-5858, 5860-5910, 5912, 5914-5923, 5925-6046 or 6047, characterized by the amino acid sequence being able to increase the efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen ), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress in a plant.

[0043] According to one aspect of some applications of the present application, an isolated polynucleotide is provided comprising a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence selected from the group consisting of the SEQ ID. No. 470-762, 3706-6046 and 6047.

[0044] According to an aspect of some applications of the present application, an isolated polynucleotide comprising a nucleic acid sequence at least 80% identical to SEQ ID is provided. No. 1-469, 763-3704 or 3705, characterized by the nucleic acid sequence being able to increase the efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, growth rate , biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress in a plant.

[0045] According to an aspect of some applications of the present application, an isolated polynucleotide comprising the nucleic acid sequence selected from the group consisting of SEQ ID is provided. No. 1-469, 763-3704 and 3705.

[0046] According to an aspect of some applications of the present patent application, a nucleic acid structure is provided, comprising the polynucleotide isolated from some applications of the present patent application and a promoter to direct the transcription of the sequence nucleic acid in a host cell.

[0047] According to an aspect of some applications of the present application, an isolated polypeptide is provided, comprising an amino acid sequence at least 80% homologous to SEQ ID. No. 470-762, 3706-5858, 5860-5910, 5912, 5914-5923, 5925-6046 or 6047, characterized by the amino acid sequence being able to increase the efficiency in the use of fertilizer (eg, efficiency in the use nitrogen content), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to plant abiotic stress.

[0048] In accordance with an aspect of some applications of the present application, an isolated polypeptide is provided, comprising the amino acid sequence selected from the group consisting of the SEQ ID. No. 470-762, 3706-6046 and 6047.

[0049] According to an aspect of some applications of the present patent application, a plant cell is provided that exogenously expresses the polynucleotide of some applications of the present patent application, or the nucleic acid structure of some applications of the present invention patent application.

[0050] According to an aspect of some applications of the present patent application, a plant cell is provided that exogenously expresses the polypeptide of some applications of the present patent application.

[0051] According to some applications of the present patent application, the nucleic acid sequence encodes an amino acid sequence selected from the group consisting of the SEQ ID. No. 470-762, 3706-6046 and 6047.

[0052] According to some applications of the present application, the nucleic acid sequence is selected from the group consisting of SEQ ID. No. 1-469, 763-3704 and 3705.

[0053] According to some applications of the present application, the polynucleotide consists of the nucleic acid sequence selected from the group consisting of the SEQ ID. No. 1-469, 7633704 and 3705.

[0054] According to some applications of the present patent application, the nucleic acid sequence encodes the selected amino acid sequence from the group consisting of the SEQ ID. No. 470-762, 3706-6046 and 6047.

[0055] According to some applications of the present patent application, the plant cell forms part of a plant.

[0056] According to some applications of the present patent application, the method further comprises the cultivation of the plant that expresses the exogenous polynucleotide under abiotic stress.

[0057] According to some applications of the present patent application, abiotic stress is selected from a group consisting of salinity, osmotic stress, drought, water deprivation, flood, etiolation, low temperature, high temperature, metal toxicity heavy, anaerobiosis, nutrient deficiency, excess nutrients, air pollution and UV irradiation.

[0058] According to some applications of the present patent application, the production comprises the production of seed or the production of oil.

[0059] In accordance with an aspect of some applications of the present patent application, a transgenic plant is provided comprising the nucleic acid structure of some applications of the present patent application.

[0060] According to some applications of the present patent application o, the method further comprises the cultivation of the plant that expresses the exogenous polynucleotide under nitrogen limiting conditions.

[0061] According to some applications of the present patent application, the promoter is heterologous to the isolated polynucleotide.

[0062] According to some applications of the present patent application, the promoter is heterologous to the host cell.

[0063] In accordance with an aspect of some applications of the present patent application, a method of cultivating a crop is provided, the method comprising sowing seeds and / or planting seedlings of a plant transformed with the polynucleotide isolated from some applications of the present patent application or the nucleic acid structure of some applications of the present patent application, characterized in that the plant is derived from plants selected from at least one characteristic selected from the group consisting of: increased efficiency in the use of nitrogen, increased tolerance to abiotic stress, increased biomass, increased growth rate, increased vigor, increased production and increased fiber production or quality and increased oil content in compared to an unprocessed plant, thereby increasing the harvest.

[0064] According to some applications of the present patent application, the untransformed plant is a wild type plant of identical genetic basis.

[0065] According to some applications of the present patent application, the untransformed plant is a wild type plant of the same species.

[0066] According to some applications of the present patent application, the unprocessed plant is grown under identical cultivation conditions.

[0067] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as that commonly understood by a person of ordinary skill in the technique to which the present application for a patent refers. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the applications of the present application for an invention patent, exemplary methods and / or materials are described below. In case of conflict, the patent's claim framework, including definitions, will prevail. In addition, the materials, methods and examples are illustrative only and are not intended to be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

[0068] Some applications of the present patent application are described here, only as examples, with reference to the accompanying drawings. Now, with specific reference to the drawings in detail, it is emphasized that the particularities shown are exemplary and for the purposes of illustrative discussion of the applications of the present application for an invention patent. In this sense, the description that accompanies the drawings makes it apparent to those skilled in the art how the applications of the present patent application can be practiced.

[0069] Figure 1 is a schematic illustration of a modified pGI binary plasmid containing the new At6669 promoter (SEQ ID No. 6052) and GUSintron (pQYN 6669) used to express the polynucleotide sequences isolated from the present patent application. invention. RB - right edge of T-DNA; LB - left edge of T-DNA; MCS - Multiple cloning site; RE-any restriction enzyme; NOS pro = nopaline synthase promoter; NPT-II = neomycin phosphotransferase gene; NOS ter = nopaline synthase terminator; Poli-A signal (polyadenylation signal); GUSintron - the GUS reporter gene (coding sequence and intron). The sequences of the polynucleotide isolated from the present application were cloned into the vector while replacing the GUSintron reporter gene.

[0070] Figure 2 is a schematic illustration of the modified binary plasmid pGI containing the new At6669 promoter (SEQ ID No. 6052) (pQFN or pQFNc) used to express the sequences of the polynucleotide isolated from the present application. RB [right border] - right edge of T-DNA; LB [left border] - left edge of T-DNA; MCS [multiple cloning site] - Multiple cloning site; RE [restriction enzyme] - any restriction enzyme; NOS pro [nopaline synthase promoter] - promoter of nopaline synthase; NPT-II [neomycin phosphotransferase] = neomycin phosphotransferase gene; NOS ter [nopaline synthase terminator] = nopaline synthase terminator; Poli-A signal (polyadenylation signal). The sequences of the polynucleotide isolated from the present application were cloned into the vector's MCS.

[0071] Figures 3A-F are images depicting the visualization of the root development of transgenic plants exogenously expressing the polynucleotide of some applications of the present patent application when grown on transparent agar plates under normal conditions (Figures 3A -B), osmotic stress (15% PEG. Figs. 3CD) or nitrogen limitation (Figures 3E-F). The different transgenes were grown on transparent agar plates for 17 days (7 days in the nursery and 10 days after transplantation). The plates were photographed every 3-4 days starting on day 1 after transplantation Fig. 3A - An image of a photograph of plants taken 10 days after transplantation on agar plates when grown under normal conditions (standard). Fig. 3B - An image of the root analysis of the plants shown in Fig. 3A in which the measured root lengths are represented by arrows. Fig. 3C - An image of a photograph of plants taken 10 days after transplantation in agar plates, grown under highly osmotic conditions (PEG 15%). Fig. 3D - An image of the root analysis of the plants shown in Fig. 3C in which the measured root lengths are represented by arrows. Fig. 3E - An image of a photograph of plants taken 10 days after transplantation on agar plates, grown under conditions of low nitrogen content. Fig. 3F - An image of the root analysis of the plants shown in Fig. 3E in which the measured root lengths are represented by arrows.

[0072] Figure 4 is a schematic illustration of the modified pGI binary plasmid containing the Root Promoter (pQNa RP, SEQ ID No. 6064) used to express the polynucleotide sequences isolated from the present application. RB - right edge of T-DNA; LB - left edge of T-DNA; NOS pro = nopaline synthase promoter; NPT-II = neomycin phosphotransferase gene; NOS ter = nopaline synthase terminator; Poli-A signal (polyadenylation signal); MCS (multiple cloning sites) of the vector used to clone the sequences of the isolated polynucleotide according to some applications of the present application.

[0073] Figure 5 is a schematic illustration of the plasmid pQYN.

[0074] Figure 6 is a schematic illustration of the plasmid pQFN.

[0075] Figure 7 is a schematic illustration of the plasmid pQFYN.

[0076] Figure 8 is a schematic illustration of the plasmid pQXNc, which is a modified binary plasmid pGI used to express the polynucleotide sequences isolated from some applications of the present application. RB - T-DNA right edge; LB - T-DNA left edge; NOS pro = nopaline synthase promoter; NPT-II neomycin phosphotransferase gene; NOS ter = nopaline synthase terminator; RE = any restriction enzyme; Poli-A signal (polyadenylation signal); 35S - the 35S promoter (pqfnc; SEQ ID No. 6048). The polynucleotide sequences isolated from some applications of the present application were cloned into the vector's MCS (multiple cloning site).

DESCRIPTION OF THE SPECIFIC APPLICATIONS

[0077] The present patent application, in some respective applications, relates to new polynucleotides and polypeptides, nucleic acid structures comprising them, host cells compressing them, transgenic plants compressing them exogenously and, more particularly, but not exclusively to methods of using them to increase efficiency in the use of nitrogen, efficiency in the use of fertilizer, production, growth rate, vigor, biomass, oil content, fiber production, fiber quality, length of fiber, tolerance to abiotic stress and / or water use efficiency of a plant.

[0078] Before explaining at least one application of the present patent application in detail, it should be understood that the present patent application is not necessarily limited by the Examples. The present invention patent application is capable of other applications or of being practiced or carried out in various ways.

[0079] The present inventors have identified new polypeptides and polynucleotides that can be used to generate nucleic acid structures, transgenic plants and to increase efficiency in the use of nitrogen, efficiency in the use of fertilizer, production, growth rate, vigor, biomass, oil content, fiber production, fiber quality, fiber length, tolerance to abiotic stress and / or water use efficiency of a plant.

[0080] Thus, as shown in the Examples section below, the present inventors used bioinformatics tools to identify polynucleotides and polypeptides that increase efficiency in the use of nitrogen, efficiency in the use of fertilizer, production (eg, production seed production, oil production), growth rate, vigor, biomass, oil content, fiber development (eg, production, length and / or quality of fiber), tolerance to abiotic stress and / or efficiency in use water from a plant. Genes that affect the trait of interest were identified (Table 1, Example 1) based on the gene expression profiles of different tissues and ecotypes of Arabidopsis, Cotton, Rice, Sorghum, Barley, Maize and Tomato (Tables 3-64; Examples 3-14) and in homology with genes known to affect the characteristic of interest and using digital expression profiles in specific tissues and conditions. Homologous polypeptides and polynucleotides (eg, orthologists) having the same function have also been identified (Table 2, Example 2). Transgenic plants overexpressing the identified polynucleotides were seen as exponents of increased biomass (eg, increased fresh and dry weight, increased leaf area, increased number of leaves, increased diameter and rosette area, increased root coverage and root length), increased production (eg, increased seed production, harvest rate and weight of 1000 seeds), increased vigor, eg, increased growth rate ( (eg, leaf area growth rate, root coverage, root length, number of leaves, diameter and area of the rosette), early flowering and inflorescence in both conditions of ideal growth (normal) and under conditions nitrogen-limiting growth factors (eg, abiotic stress condition, such as nutrient deficiency stress) (Tables 66-93; Examples 18-20). Taken together, these results suggest the use of the new polynucleotides and polypeptides of the present patent application to increase efficiency in the use of nitrogen, efficiency in the use of fertilizer, production (eg, seed production, oil production) , growth rate, vigor, biomass, oil content, fiber production, fiber quality, fiber length, tolerance to abiotic stress and / or water use efficiency of a plant.

[0081] Thus, according to an aspect of some applications of the present patent application, a method is provided to increase efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence encoding at least one polypeptide about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86% at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92% at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or, say, 100% homologous to the amino acid sequence selected from the group consisting of the SEQ ID. N ° 470-762, 3706-5858, 5860-5910, 5912, 5914-5923, 5925-6046 and 6047, thus increasing the efficiency in the use of fertilizer (eg efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress in a plant.

[0082] As used here, the term "efficiency in the use of fertilizer" refers to the metabolic process (es) that leads to an increase in plant production, biomass, vigor and rate of growth per unit of fertilizer applied. The metabolic process can be the capture, propagation, absorption, accumulation, reallocation (within the plant) and use of one or more minerals and organic portions absorbed by the plant, such as nitrogen, phosphates and / or potassium.

[0083] As used herein, the term "limiting fertilizer conditions" refers to growing conditions that include a level (eg, concentration) of an applied fertilizer that is below the level required for normal plant metabolism , growth, reproduction and / or viability.

[0084] As used here, the expression "efficiency in the use of nitrogen (NUE)" refers to the metabolic process (s) that leads to an increase in plant production, biomass, vigor and growth rate per unit of nitrogen applied. The metabolic process can be uptake, propagation, absorption, accumulation, reallocation (within the plant) and use of nitrogen absorbed by the plant.

[0085] As used herein, the term "nitrogen limiting conditions" refers to growth conditions that include a level (eg, concentration) of applied nitrogen (ie, ammonia or nitrate) that is below the level necessary for normal plant metabolism, growth, reproduction and / or viability.

[0086] The improved NUE and FUE of the plant are translated into the field in similar amounts of crop yield, while implementing less fertilizers, or improved yields acquired by implementing the same levels of fertilizers. Thus, improved NUE or FUE has a direct effect on plant production in the field. Thus, the polynucleotides and polypeptides of some applications of the present patent application positively affect plant production, seed production and plant biomass. In addition, the benefit of improved plant NUE will certainly improve the quality of the crop and biochemical constituents of the seed such as protein production and oil production.

[0087] As used here, the term "plant production" refers to the amount (eg, as determined by weight or size) or quantity (numbers) of tissues or organs produced by plants or by growing season. Therefore, the increase in seed production per plant can affect the economic benefit that someone can obtain from the plant in a given growing area and / or growing season.

[0088] It is important to note that the production of the plant can be affected by several parameters, including, but not limited to, the biomass of the plant; plant vigor; growth rate; seed production; amount of seeds or grains; seed or grain quality; oil production; oil, starch and / or protein content in harvested organs (eg, seeds or plant parts of the plant); number of flowers (florets) per panicle (expressed as a proportion of the number of seeds filled over the number of primary panicles); harvest index; number of plants grown per area; number and size of organs harvested per plant and area; number of plants per growth area (density); number of organs harvested in the field; total leaf area; carbon assimilation and carbon partitioning (the distribution / allocation of carbon within the plant); shadow resistance; number of collectable organs (for example, seeds), seeds per pod, weight per seed; and modified architecture [such as increasing stem diameter, thickness, or improving physical properties (for example, elasticity)].

[0089] As used here, the term "seed production" refers to the number or weight of seeds per plant, seeds per pod or area of cultivation or the weight of a single seed, or the oil extracted per seed. Therefore, seed production can be affected by the dimensions of the seed (eg, length, width, perimeter, area and / or volume), the number of seeds (full) and the rate of seed filling and the content of seed oil. Therefore, the increase in seed production per plant can affect the economic benefit that someone can obtain from the plant in a given growing area and / or growing season; and increasing seed production per cultivation area can be achieved by increasing seed production per plant, and / or increasing the number of plants grown in a given area.

[0090] The term "seed" (also referred to as "grain" or "nucleus"), as used here, refers to a small embryonic plant confined to a cover called a seed coat (usually with some stored food), the product of the matured ovum of gymnosperm and angiosperm plants that occurs after fertilization and some growth within the mother plant.

[0091] The term "oil content", as used here, refers to the amount of lipids in a given plant organ, whether the seeds (seed oil content) or the plant portion of the plant (vegetable oil content ) and is expressed as a percentage of dry weight (10% of seed moisture) or wet weight (for the vegetable portion).

[0092] It should be noted that the oil content is affected by the intrinsic oil production of a tissue (eg, seed, plant part), as well as the mass or size of the oil-producing tissue per plant or per growth period.

[0093] In an application, the increase in the oil content of the plant can be achieved by increasing the size / mass of tissue (s) of a plant, which comprises the oil by growing period. In this way, increasing the oil content of a plant can be achieved by increasing the production, the growth rate, the biomass and the vigor of the plant.

[0094] As used here, the term "plant biomass" refers to the amount (eg, measured in grams of tissue dried by the air) of a tissue produced from the plant in a growing period, which also it can determine or affect plant production or production by cultivation area. An increase in plant biomass can occur in the whole plant or in parts of it as parts above ground level (harvestable), plant biomass, roots and seeds.

[0095] As used here, the term "growth rate" refers to the increase in the size of the organ / tissue of the plant per period (can be measured in cm2 per day).

[0096] As used here, the expression "plant vigor" refers to the quantity (measured by weight) or tissue produced by the plant in a given period. Therefore, the increase in vigor can determine or affect the production of the plant or the production by period of cultivation or by area of cultivation. In addition, premature vigor (seed and / or seedling) results in a better position in the field.

[0097] Improving early vigor is an important goal of modern rice breeding programs in temperate and tropical rice cultivars. Long roots are important for adequate soil fixation in pre-germinated rice. When rice is directly sown in flooded fields and where the plants are expected to emerge quickly through the water, longer shoots are associated with vigor. Where drilling sowing is practiced, longer mesocotyl and coleoptile are important for good seedling emergence. The ability to project early vigor on plants would be of great importance in agriculture. Eg, low early vigor has been a limitation in the introduction of hybrid maize (Zea mays L.) based on the corn belt germplasm in the European Atlantic.

[0098] It should be noted that plant production can be determined under stress (eg, abiotic stress, nitrogen limiting conditions) and / or non-stress conditions (normal).

[0099] As used here, the term "non-stress conditions" refers to growing conditions (eg, water, temperature, light-dark cycles, humidity, salt concentration, concentration of fertilizer in the soil, supply nutrients such as nitrogen, phosphorus and / or potassium), which do not significantly go beyond the daily climatic and other abiotic conditions that plants may encounter and which allow the ideal growth, metabolism, reproduction and / or viability of a plant at any stage in its life cycle (eg, from a seed crop plant to a mature plant and back to the seed again). Technicians in the field are aware of the normal soil and climatic conditions for a given plant in a given geographic location. It should be noted that while non-stress conditions may include some mild variations from ideal conditions (which vary from one type / species from one plant to another), such variations do not cause the plant to stop growing without the ability to resume growth. growth.

[00100] The expression "abiotic stress", as used here, refers to any adverse effect on the metabolism, growth, reproduction and / or viability of the plant. Consequently, abiotic stress can be induced by suboptimal environmental growth conditions such as, for example, salinity, water deprivation, flooding, freezing, low or high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, air pollution or UV irradiation. The implications of abiotic stress are discussed in the History section.

[00101] The term "tolerance to abiotic stress", as used here, refers to a plant's ability to withstand abiotic stress without undergoing a substantial change in metabolism, growth, productivity and / or viability.

[00102] Plants are subjected to a range of environmental challenges. Several of these, including saline stress, general osmotic stress, arid stress and freezing stress, have the ability to impact total plant and cellular water availability. Not surprisingly, then, the plant's responses to this collection of stresses are related. Zhu (2002) Ann. Rev. Planta Biol. 53: 247-273 et al. Note that "most studies on water stress signaling have focused on salt stress primarily because the plant's responses to salt and drought are closely related and the mechanisms overlap." Many examples of similar responses and paths to this set of stresses have been documented. For example, CBF transcription factors demonstrated conditioning of salt resistance, freezing and drought (Kasuga et al. (1999) Nature Biotech. 17: 287-291). The Arabidopsis rd29B gene is induced in response to salt stress and dehydration, a process that is largely mediated through an ABA signal transduction process (Uno et al. (2000) Proc. Natl. Acad. Sci. USA 97: 11632 -11637), resulting in altered activity of transcription factors that bind to an upstream element within the rd29B promoter. In Mesembryanthemum crystallinum (ice plant), Patharker and Cushman showed that a calcium-dependent protein kinase (McCDPK1) is induced by exposure to drought and salt stresses (Patharker and Cushman (2000) Plant J. 24: 679-691). Stress-induced kinase has also been shown to phosphorylate a transcription factor, probably altering its activity, although the transcribed levels of the target transcription factor are not altered in response to salt stress or drought. Similarly, Saijo et al. demonstrated that a salt-induced / drought-induced kalodulin-dependent protein kinase (OsCDPK7) conferred high salt tolerance and drought for rice when overexpressed (Saijo et al. (2000) Planta J. 23: 319-327).

[00103] Dehydration exposure evokes similar survival strategies in plants, as does freezing stress (see, eg, Yelenosky (1989) Planta Physiol 89: 444-451) and drought stress induces freezing tolerance (see, eg, Siminovitch et al. (1982) Planta Physiol 69: 250-255; and Guy et al. (1992) Plant 188: 265-270). In addition to inducing cold acclimatization proteins, strategies that allow plants to survive in low water conditions may include, for example, reduced surface area, or surface oil or wax production. In another example, the high solute content of the plant prevents evaporation and water loss due to heat, drought, salinity, osmotic and the like, thus providing better tolerance to the stresses above.

[00104] It will be understood that some pathways involved in resistance to stress (as described above), will also be involved in resistance to other stresses, regulated by the same genes or counterparts. Certainly, resistance paths are related, not identical, and therefore, not all genes that control resistance to stress will control resistance to other stresses. Nevertheless, if a resistance of gene conditions to one of these stresses would be evident to the person skilled in the art, the test for resistance to these related stresses. Methods for assessing stress resistance are also provided in the Examples section below.

[00105] As used here, the expression "water use efficiency (WUE)" refers to the level of organic matter produced per unit of water consumed by the plant, that is, the dry weight of a plant in relation to the use of plant water, eg biomass produced by unit transpiration.

[00106] It should be noted that an improved ABST will give plants improved vigor also in non-stress conditions, resulting in crops that have improved biomass and / or production, eg, elongated fibers for the cotton industry, content of higher oil.

[00107] The term "fiber" is usually inclusive of thick-walled conduction cells, such as vessels and tracheids, and to fibrillar aggregates of many individual fiber cells. Therefore, the term "fiber" refers to (a) cells that conduct and do not conduct a thick wall of the xylem; (b) fibers of extraxylary origin, including those of phloem, bark, soil tissue and epidermis; and (c) fibers from the stems, leaves, roots, seeds and flowers or inflorescences (such as those from Sorghum vulgare used in the manufacture of brushes and brooms).

[00108] Examples of plant that produces fiber, include, among others, agricultural crops such as cotton, cotton silk tree (Paina, Ceiba pentandra), desert willow, creosote bush, winterfat, balsa, quenafe, rosala, jute , abaca sisal, flax, corn, sugar cane, hemp, ramie, paina, coconut fiber, bamboo, Spanish moss and Agave spp. (e.g., sisal).

[00109] As used here, the term "fiber quality" refers to at least one fiber parameter that is agriculturally desired, or required in the fiber industry (also described below). Examples of such parameters include, but are not limited to, fiber length, fiber strength, fiber suitability, fiber weight per unit length, maturity ratio and uniformity (also described below).

[00110] The quality of the cotton fiber (gauze) is typically measured according to the length, strength and delicacy of the fiber. Consequently, the quality of the gauze is considered to be higher when the fiber is longer, stronger and thinner.

[00111] As used here, the term "fiber production" refers to the quantity or quality of fibers produced from the fiber-producing plant.

[00112] As used herein, the term "increase" refers to at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% increase in efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, growth rate , biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress of a plant compared to a native plant or wild type plant (ie, a plant not modified with the biomolecules (polynucleotide or polypeptides) of the present patent application, eg, an unprocessed plant of the same species that is grown under the same (eg, identical) growing conditions].

[00113] The expression "express an exogenous polynucleotide within the plant", as used here, refers to the upward regulation of the level of expression of an exogenous polynucleotide within the plant, introducing the exogenous polynucleotide into a plant or plant cell and expressing it by recombinant means, as also described below.

[00114] As used here, "express" refers to expression in the mRNA and, optionally, at the polypeptide level.

[00115] As used herein, the term "exogenous polynucleotide" refers to a heterologous nucleic acid sequence that may not be naturally expressed within the plant (e.g., a nucleic acid sequence of different species) or in which overexpression in the plant is desired. The exogenous polynucleotide can be introduced into the plant in a stable or transient manner, in order to produce a ribonucleic acid molecule (RNAIribonucleic acid) and / or a polypeptide molecule. It should be noted that the exogenous polynucleotide can comprise a nucleic acid sequence that is identical or partially homologous to a plant endogenous nucleic acid sequence.

[00116] The term "endogenous", as used here, refers to any polynucleotide or polypeptide that is present and / or naturally expressed within a plant or a cell thereof.

[00117] According to some applications of the present patent application, the exogenous polynucleotide of the present patent application comprises a nucleic acid sequence that encodes a polypeptide having an amino acid sequence of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least , about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about at least 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 %, or, say, 100% homologous to the amino acid sequence selected from the group consists of SEQ IDs. No. 470-762, 3706-5858, 58605910, 5912, 5914-5923, 5925-6046 and 6047.

[00118] Homologous sequences include both orthologous and parallel sequences. The term "paralog" refers to gene duplications within the genome of a species leading to paralog genes. The term "orthologist" refers to the homologous genes in different organisms due to the ancestral relationship.

[00119] An option to identify orthologists in monocotyledonous plant species is to conduct a reciprocal explosion search. This can be done through a first explosion, involving exploding the sequence of interest against any database in the sequence, such as the publicly available NCBI database which can be found at: http: // www [Http: // World Wide Web 1 Hypertext Transfer Protocol: World Wide Web / Internet] (dot) ncbi (dot) nlm (dot) nih (dot) gov. If orthologists are sought in the rice, the sequence of interest would be detonated again, eg, the 28,469 full-length cDNA clones from Oryza Sativa Nipponbare available from NCBI. The results of the explosion can be filtered. The full-length sequences of the filtered or unfiltered results are then detonated back (second explosion) against the organism sequences from which the sequence of interest is derived. The results of the first and second explosions are then compared. An orthologist is identified when the sequence resulting in the highest score (best hit) in the first explosion identifies in the second explosion the query sequence (the original interest sequence) as the best hit. Using the same rationale, a parallel (homologous to a gene in the same organism) is found. In the case of large sequence families, the ClustalW program can be used [http: // www (dot) ebi (dot) ac (dot) uk / Tools / clustalw2 / index (dot) html], followed by a union tree neighbor (http: // en (dot) wikipedia (dot) org / wiki / Neighbor-joining) that helps in visualizing the grouping.

[00120] Homology (eg homology percentage, identity + similarity) can be determined using any homology comparison software that calculates the sequence alignment in pairs.

[00121] Identity (eg, homology percentage) can be determined using any homology comparison software, including, for example, BlastN software or BlastP from the National Center of Biotechnology Information [NCBI ❑ National Information Center Biotechnology], as well as using standard parameters.

[00122] According to some applications of the present patent application, the identity is a global identity, that is, an identity on the entire amino acid or nucleic acid sequence of the present patent application and not only on parts respective.

[00123] According to some applications of the present patent application, the term "homology" or "homologue" refers to the identity of two or more nucleic acid sequences; or identity of two or more amino acid sequences; or the identity of an amino acid sequence to one or more nucleic acid sequences encoding the same.

[00124] According to some applications of the present patent application, the homology is a global homology, that is, a homology over the entire amino acid or nucleic acid sequence of the present patent application and not just parts respective.

[00125] The degree of homology or identity between two or more sequences can be determined using various tools of comparison of known sequences. In the sequence there is a non-limiting description of such tools, which can be used together with some applications of the present patent application.

[00126] Global alignment in pairs was defined by SB Needleman and CD Wunsch, "A general method applicable to the search of similarities in the amino acid sequence of two proteins" Journal of Molecular Biology, 1970, pages 443-53, volume 48) .

[00127] For example, when starting with a polypeptide sequence and comparing with other polypeptide sequences, the EMBOSS-6.0.1 algorithm from Needleman-Wunsch (available at http: // emboss (dot) sourceforge (dot) net / apps / cvs / emboss / ap ps / needle (dot) html) can be used to find the ideal alignment (including the gaps) of two strings together with their total lengths - a "global alignment". Standard parameters for the Needleman-Wunsch algorithm (EMBOSS-6.0.1) include: gapopen = 10; gapextend = 0.5; datafile = EBLOSUM62; brief = YES.

[00128] According to some applications of the present patent application, the parameters used with the EMBOSS-6.0.1 tool (for protein-protein comparison) include: gapopen = 8; gapextend = 2; datafile = EBLOSUM62; brief = YES.

[00129] According to some applications of the present patent application, the limit used to determine homology using the EMBOSS-6.0.1 algorithm from Needleman-Wunsch is 80%, 81%, 82%, 83%, 84% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[00130] When starting with a polypeptide sequence and comparing polynucleotide sequences, the OneModel FramePlus algorithm (Halperin, E., Faigler, S. And Gill-More, R. (1999) - FramePlus: aligning DNA to protein sequences. Bioinformatics, 15, 867-873) (available at http: // www (dot) biocceleration (dot) com / Products (dot) html) can be used with the following standard parameters: model = frametp2n.model mode = local.

[00131] According to some applications of the present patent application, the parameters used with the OneModel FramePlus algorithm are model = frame + p2n.model, mode = qglobal.

[00132] According to some applications of the present patent application, the limit used to determine homology using the OneModel FramePlus algorithm is 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[00133] When starting with a polynucleotide sequence and comparing with other polynucleotide sequences, the EMBOSS-6.0.1 algorithm from NeedlemanWunsch (available at http: // emboss (dot) sourceforge (dot) net / apps / cvs / emboss / ap ps / needle (dot) html) can be used with the following standard parameters. (EMBOSS-6.0.1) gapopen = 10; gapextend = 0.5; datafile = EDNAFULL; brief = YES.

[00134] According to some applications of the present patent application, the parameters used with the EMBOSS-6.0.1 algorithm of NeedlemanWunsch are gapopen = 10; gapextend = 0.2; datafile = EDNAFULL; brief = YES.

[00135] According to some applications of the present patent application, the limit used to determine homology using the Needleman-Wunsch EMBOSS-6.0.1 algorithm for the comparison of polynucleotides with polynucleotides is 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[00136] According to some applications, the determination of the degree of homology additionally requires the use of the SmithWaterman algorithm (for protein-protein comparison or nucleotide-nucleotide comparison).

[00137] Standard parameters for the GenCore 6.0 SmithWaterman algorithm include: model = sw.model.

[00138] According to some applications of the present patent application, the limit used to determine homology using the Smith-Waterman algorithm is 80%, 81%, 82%, 83%, 84%, 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[00139] According to some applications of the present patent application, the global homology is performed on the sequences pre-selected by local homology for the polypeptide or polynucleotide of interest (e.g., 60% identity over 60% of the length of the sequence) before performing the overall homology for the polypeptide or polynucleotide of interest (e.g., 80% overall homology over the entire sequence). For example, homologous sequences are selected using the BLAST software with the Blastp and tBlastn algorithms acting as filters for the first stage and the needle (EMBOSS package) or the Structure + algorithmic alignment for the second stage. The local identity (Blast alignments) is defined with a very permissive cut - 60% of Identity in a range of 60% of the sequence lengths because it uses only one filter for the global alignment phase. The standard filtering of the Blast package was not used (setting the parameter "-F F").

[00140] In the second stage, homologues are defined based on the global identity of at least 80% for the main polypeptide sequence.

[00141] According to some applications of the present patent application, two different ways for discovering the ideal global alignment for the protein or nucleotide sequences are used:

[00142] Between two proteins (following the blastp filter): Needleman-Wunsch's EMBOSS-6.0.1 algorithm with the following modified parameters: gapopen = 8 gapextend = 2. The rest of the parameters remain unchanged from the standard options listed here:

Standard qualifiers (Required): [-asequence] string File name of the string and optional format, or reference (US entry).

[00143] [-bsequence] seciallFile name of the sequence (s) and optional format, or reference (US entry). -gapopenflutuante [10.0 for any sequence]. The open gap penalty is a classification taken when a gap is created. The best value depends on the choice of the comparison matrix. The default value assumes that you are using the EBLOSUM62 matrix for the protein sequences and the EDNAFULL matrix for the nucleotide sequences.

[00144] (floating point number from 1.0 to 100.0)

[00145] gapextend floating 0.5 for any sequence]. The penalty for extending the gap is added to the standard gap penalty for each base or residue in the gap. This is the time that the gap is penalized. Normally, few large gaps are expected and not many small gaps, so the penalty for the length of the gap should be less than the penalty for the gap. An exception is when one or more strings are read alone with possible string errors, in which case you would expect many simple base gaps. This result can be achieved by setting the open gap penalty to zero (or very low) and using the gap extension penalty to control the gap rating. (floating point number from 0.0 to 10.0). [-outfile] alignment [* .needle] Name of the output alignment file

Additional Qualifiers (Optional): datafile matrixf [EBLOSUM62 for protein, EDNAFULL for DNA]

[00146] This is a classification arrangement file used when comparing the strings. By default, this is the `EBLOSUM62 'file (for proteins) or the' EDNAFULL 'file (for nucleic sequences) These files are found in the` data' directory of the EMBOSS installation.

Advanced Qualifiers (Spontaneous):

[00147] [no] Boolean brief [Y] Brief identity and similarity.

Associated Qualifiers:

[00148] "-asequence" Associated Qualifiers.

[00149] sbeginl integer Starts the sequence to be used.

[00150] -sendl integer End the sequence to be used.

[00151] Reverse Boolean sreversel (if DNA).

[00152] boolean saskl Asks to start / end / revert.

[00153] boolean snucleotidel The sequence is nucleotide.

[00154] boolean sproteínal The sequence is protein.

[00155] -slowerl boolean Makes lowercase.

[00156] boolean supperl Capitalize.

[00157] -sformatl string Input string format.

[00158] sdbnamel string Name of the database.

[00159] -sidl string Name of the entry.

[00160] ufol Resources UFO chain.

[00161] fformatl string Resource Format.

[00162] fopenfilel string File name of the resources.

[00163] "-bsequence" Associated Qualifiers.

[00164] sbegin2 integer Starts each sequence to be used.

[00165] send2 integer End each sequence to be used.

[00166] reverse boolean sreverse2 (if DNA).

[00167] sask2 boolean Asks to start / end / revert.

[00168] boolean -snucleotide2 The sequence is nucleotide.

[00169] boolean sproteina2 The sequence is protein.

[00170] boolean slower2 Makes lowercase.

[00171] boolean supper2 Capitalize.

[00172] sformat2 string Input string format.

[00173] -sdbname2 string Name of the database.

[00174] sid2 string Name of the entry.

[00175] ufo2 UFO Resources chain.

[00176] fformat2 string Resource Format.

[00177] -fopenfile2 string File name of the resources.

[00178] "-outfile" Associated Qualifiers.

[00179] aformat3 string Format of Alignments.

[00180] -aextension3 string Extension of the file name.

[00181] -adirectory3 string Output Directory.

[00182] aname3 string Name of the base file.

[00183] awidth3 integer Width of alignment.

[00184] aaccshow3 boolean Shows the access number in the header.

[00185] adesshow3 boolean Shows the description in the header.

[00186] ausashow3 boolean Shows the complete USA in alignment.

[00187] boolean aglobal3 Shows the complete sequence in the alignment.

General Qualifiers:

[00188] auto boolean Turn off requests.

[00189] boolean stdout Writes the first file to standard output.

[00190] - Boolean filter Reads the first file from standard input, writes the first file to standard output.

[00191] Boolean options Prompts for standard and additional values.

[00192] debug boolean Writes the debug output of the .dbg program

[00193] Boolean verbose Registers some / all command line options.

[00194] Boolean help Registers command line options.

[00195] More information about the qualifiers:

[00196] associated and general can be found with -

[00197] help -verbose.

[00198] -warning Boolean Records warnings.

[00199] - Boolean error Records errors.

[00200] fatal boolean Records fatal errors.

[00201] die boolean Records the expiration messages of the program.

[00202] Between a protein sequence and a nucleotide sequence (following the rblastn filter) Application of GenCore 6.0 OneModel using the structure + algorithm with the following parameters: model = frame + p2n.model mode = qglobal -q = protein.sequence - db = nucleotide.sequence. The rest of the parameters remain unchanged from the standard options:

[00203] Use:
om -model = <model fname> [- q =] query [-db =] database [options].

[00204] model = <model fname> Specifies the model you want to run. All models provided by Compugen are located in the $ CGNROOT / models / directory.

[00205] Valid command line parameters:
dev = <dev name> Selects the device to be used by the application.

[00206] The valid devices are:
bic - Bioccelerator (valid for SW, XSW, FRAMEN2P, and FRAME P2N models).

[00207] xlg - BioXL / G (valid for all models, except XSW). xlp - BioXL / P (valid for SW, FRAME + N2P, and
FRAME P2N models).

[00208] xlp - BioXL / H (valid for SW, FRAME + N2P, and
FRAME P2N models).

[00209] soft - Software device (for all models). - q = <query> Defines the set of queries. The queries can be a sequence file or a reference to the database.

[00210] You can specify a query by name or by access number. The format is detected automatically.

[00211] However, you can specify a format using the -qfmt parameter

[00212] If a query is not specified, the program requests one.

[00213] If the query set is a reference to the database, an output file is produced for each sequence in the query.

[00214] -db = <database name> Choose the database set. The database set can be a sequence file or a reference to the database. The database format is detected automatically. However, you can specify a format using the -dfmt parameter.

[00215] qacc Adds this parameter to the command line if a query is specified using the access numbers.

[00216] dacc Adds this parameter to the command line if a database is specified using access numbers.

[00217] dfmt / -qfmt = <formattype> Choose the type of database / query format.

[00218] The possible formats are:
fasta - fasta with the type of autodetected sequence. fastap - fasta protein sequence.

[00219] fastan - fasta nucleic sequence.

[00220] gcg - gcg format, the type is autodetected.

[00221] gcg - gcg format, the type is autodetected.

[00222] gcg9seqp - protein sequence of gcg9 format.

[00223] gcg9seqn - nucleic sequence of gcg9 format.

[00224] nbrf - nbrf sequence, the type is autodetected.

[00225] nbrfp - nbrf protein sequence.

[00226] nbrfn - nucleic sequence nbrf.

[00227] embl - embl and swissprot format.

[00228] genbank - genbank (nucleic) format.

[00229] blast - blast format.

[00230] nbrf gcg - nbrf gcg sequence, the type is autodetected. nbrf gcgp - nbrf-gcg protein sequence.

[00231] nbrfgcgn - nucleic sequence nbrf-gcg.

[00232] raw - raw ascii sequence, the type is autodetected.

[00233] rawp - raw ascii protein sequence.

[00234] rawn - raw ascii nucleic sequence.

[00235] pir - pir codata format, the type is autodetected.

[00236] Profile - gcg profile (valid only for -qfmt

[00237] in SW, XSW, FRAME P2N and FRAME + P2N).

[00238] out = <out fname> The name of the output file.

[00239] suffix = <name> The suffix of the output file name.

[00240] gapop = <n> Penalty for opening a gap. This parameter is not valid for FRAME +.

[00241] For FrameSearch the default is 12.0. For other surveys the default is 10.0.

[00242] gapext = <n> Penalty for extension of gap. This parameter is not valid for FRAME +.

[00243] For FrameSearch the default is 4.0. For other models: the standard for protein searches is 0.05 and the standard for nucleic searches is 1.0.

[00244] ggapop = <n> The penalty for opening a gap following consultations. The default is 10.0. Valid for XSW.

[00245] ggapext = <n> The penalty for the extension of a gap following questions. The default is 0.05. Valid for XSW.

[00246] start = <n> The position in the query string to start the search.

[00247] end = <n> The position in the query string to stop the search.

[00248] qtrans Performs a translated search, relevant to a nucleic query against a protein database.

[00249] The nucleic query is translated into six reading structures and a result is provided for each structure. Valid for SW and XSW.

[00250] Note: the options "-qtrans" and "-dtrans" are mutually exclusive.

[00251] matrix = <matrix file> Specifies the comparison matrix to be used in the search. The matrix must be in BLAST format. If the matrix file is not found in $ CGNROOT / tables / matrix, specify the full path as the value for the -matrix parameter.

[00252] trans = <transtab name> Translation table. The default location for the table is $ CGNROOT / tables / trans.

[00253] onestrand Restricts the search to only the top strand of the query / database of the nucleic sequence.

[00254] list = <n> The maximum size of the output hit list.

[00255] The default is 50.

[00256] docalign = <n> The number of lines of documentation preceding each alignment. The default is 10.

[00257] -thr score = <score name> The classification that places a limit on the display of results. Ratings less than the minimum value of -thr min or greater than the value -thr max are not shown. Valid options are:
quality.

[00258] Score.

[00259] Score.

[00260] thr max = <n> The highest rating limit. Results greater than the -thr max value are not shown. -thr min = The lowest rating limit. Results less than the -thrmin value are not shown.

[00261] -align = <n> The number of alignments registered in the output file.

[00262] -noalign Does not display the alignment.

[00263] Note: the parameters "-align" and "-noalign" are mutually exclusive.

[00264] -outfmt = <format name> Specifies the type of output format. The default format is PFS. Possible values are:
PFS - PFS text format
FASTA - text format FASTA
BLAST - BLAST text format

[00265] nonorm Does not normalize the classification.

[00266] norm = <norm name> Specify the normalization method. Valid options are:
log - normalization of the algorithm.

[00267] std - standard normalization.

[00268] stat - Statistical method of Ervilharson

[00269] Note: the parameters "-nonorm" and "-norm" cannot be used together.

[00270] Note: Parameters -xgapop, -xgapext, -fgapop, -fgapext, - ygapop, -ygapext, -delop and -delext apply only to FRAME +.

[00271] xgapop = <n> The penalty for opening a gap when inserting a codon (triple). The default is 12.0.

[00272] -xgapext = <n> The penalty for the extension of a gap when inserting a codon (triple). The default is 4.0.

[00273] ygapop = <n> The penalty for opening a gap when excluding an amino acid. The default is 12.0.

[00274] -ygapext = <n> The penalty for extending a gap when excluding an amino acid. The default is 4.0.

[00275] -fgapop = <n> The penalty for opening a gap when inserting a base of DNA. The default is 6.0.

[00276] fgapext = <n> The penalty for the extension of a gap when inserting a base of DNA. The default is 7.0.

[00277] delop = <n> The penalty for opening a gap when excluding a base of DNA. The default is 6.0.

[00278] delext = <n> The penalty for the extension of a gap when excluding a base of DNA. The default is 7.0.

[00279] -silent No output for the screen is produced.

[00280] host = <host name> 0 name of the organizer on which the server works. By default, the application uses a specific organizer in the $ CGNROOT / cgnhosts file.

[00281] wait Do not access the background when the device is busy. This option is not relevant for the Parseq or Soft pseudodevice.

[00282] -batch Run the job in the background. When this option is specified, the file "$ CGNROOT / defaults / batch.defaults"

[00283] is used to choose the batch command. If the file does not exist, the command "at now" is used to perform the job.

[00284] Note: the parameters "-batch" and "-wait" are mutually exclusive.

[00285] -version Prints the version number of the software.

[00286] -help Displays this help message. For more specific help type: "om -model = <model fname> -help".

[00287] According to some applications, homology is a local homology or a local identity.

[00288] Local algorithm tools include, but are not limited to, BlastP, BlastN, BlastX or TBLASTN software from the National Biotechnology Information Center (NCBI), FASTA and the Smith-Waterman algorithm.

[00289] A tblastn search allows the comparison between a protein sequence and translations of six structures in a nucleotide database. It can be a very productive way to find encoded regions of homologous protein in unnotated nucleotide sequences, such as [EST's ❑ expressed sequence tags] and genome record projects [HTG ❑ draft genome records], located in BLAST est and htgs databases, respectively.

[00290] Standard parameters for blastp include: Maximum target strings: 100; Expected limit: e-5; Word size: 3: Maximum matches in a query range: 0; Classification parameters: Matrix - BLOSUM62; filters and camouflage: Filter - regions of low complexity.

[00291] Local alignment tools that can be used include, but are not limited to, the tBLASTX algorithm, which compares the conceptual translation products of six structures of a sequence of nucleotide queries (both strands) against a sequence database of protein. Standard parameters include: Maximum target strings: 100; Expected limit: 10; Word size: 3: Maximum matches in a query range: 0; Classification parameters: Matrix - BLOSUM62; filters and camouflage: Filter - regions of low complexity.

[00292] According to some applications of the present patent application, the exogenous polynucleotide of the present patent application encodes a polypeptide having an amino acid sequence of at least 80%, at least 81%, at least 82 %, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, say, 100% identical to the selected amino acid sequence from the consistent group of SEQ ID No.: 470-762, 37065858, 25 5860-5910, 5912, 5914-5923, 5925- 6046 and 6047.

[00293] According to some applications of the present patent application, the method for increasing the efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, growth rate, biomass , vigor, fiber production, fiber quality and / or tolerance to a plant's abiotic stress is effected by expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence that encodes a polypeptide of at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least , about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about at least 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99 %, or, say, 100% identical to the amino acid sequence selected from the group consisting of SEQ IDs. N ° 470-762, 3706-5858, 5860-5910, 5912, 5 5914- 5923, 5925-6046 and 6047, thereby increasing efficiency in the use of fertilizer (eg efficiency in the use of nitrogen) , oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to plant abiotic stress.

[00294] According to some applications of the present application, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence established by SEQ ID. 470762, 3706-6046 or 6047.

[00295] According to an aspect of some applications of the present patent application, the method for increasing the efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, rate of growth, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress of a plant is effected by expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence that encodes a polypeptide comprising an amino acid sequence selected from the group consisting of the SEQ ID. N ° 470-762, 3706-6046 and 6047, thereby increasing efficiency in the use of fertilizer (eg efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to plant abiotic stress.

[00296] According to an aspect of some applications of the present application, a method is provided to increase efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence that encodes a polypeptide selected from the group consisting of ID SEQ. N ° 470-762, 3706- 6046 and 6047, thus increasing efficiency in the use of fertilizer (eg efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to plant abiotic stress.

[00297] According to some applications of the present application, the exogenous polynucleotide encodes a polypeptide consisting of the amino acid sequence established by SEQ ID. No. 470-762, 3706-6046 and 6047.

[00298] According to some applications of the present application, the exogenous polynucleotide comprises a nucleic acid sequence that is at least about 80%, at least about 81%, at least about 82 at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least , about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, e.g. , 100% identical to the nucleic acid sequence selected from the group consisting of SEQ IDs. No. 1-469, 763-3704 and 3705.

[00299] In accordance with an aspect of some applications of the present application for a patent, a method is provided to increase efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress of a plant, comprising expressing within the plant an exogenous polynucleotide comprising a nucleic acid sequence of at least about 80%, at least at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93% at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% , P less than about 99%, e.g., 100% identical nucleic acid sequence selected from the group consisting of SEQ ID. N ° 1-469, 7633704 and 3705, thus increasing efficiency in the use of fertilizer (eg efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, production of fiber, fiber quality and / or tolerance to plant abiotic stress.

[00300] According to some applications of the present application, the exogenous polynucleotide is at least about 80%, at least about 81%, at least about 82%, at least about 83% at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89% at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, eg 100% identical to the selected polynucleotide of the group consisting of the SEQ ID. No. 1-469, 763-3704 and 3705.

[00301] According to some applications of the present patent application, the exogenous polynucleotide is established by SEQ ID. No. 1469, 763-3704 or 3705.

[00302] As used herein, the term "polynucleotide" refers to a single or double stranded nucleic acid sequence that is isolated and supplied in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a sequence polynucleotide genomics and / or a composite polynucleotide sequence (eg, a combination of the above sequences).

[00303] The term "isolated" refers to at least partially separated (a) from the natural environment, e.g., from a plant cell.

[00304] As used herein, the term "complementary polynucleotide sequence" refers to a sequence that results from the reverse transcription of messenger RNA using reverse transcriptase or any other RNA-dependent DNA polymerase. This sequence can be subsequently amplified in vivo or in vitro using a DNA-dependent DNA polymerase.

[00305] As used herein, the term "polynucleotide genomic sequence" refers to a sequence derived (isolated) from a chromosome and thus represents a contiguous portion of a chromosome.

[00306] As used herein, the term "compound polynucleotide sequence" refers to a sequence that is at least partially complementary and at least partially genomic. The composite sequence may include some exonal sequences necessary to encode the polypeptide of the present application, as well as some intronic sequences interposed with each other. Intronic sequences can be from any source, including other genes, and will typically include preserved interlaced signal sequences. These intronic sequences may also include elements that regulate the expression cisatuantes.

The nucleic acid sequences encoding the polypeptides of the present application can be optimized for expression. Examples of these sequence modifications include, but are not limited to, an altered G / C content of a more careful approach than that commonly found in the plant species of interest and the removal of atypical codons found in plant species commonly called optimization. of codons.

[00308] The term "codon optimization" refers to the selection of DNA nucleotides appropriate for use within a structural gene or fragment thereof that addresses the use of the codon within the plant of interest. Therefore, an optimized gene or nucleic acid sequence refers to a gene in which the nucleotide sequence of a native or naturally occurring gene has been modified to use statistically preferred or statistically favored codons within the plant. Typically, the nucleotide sequence is examined at the DNA level and in the coding region optimized for expression in the determined plant species using any suitable procedure, e.g., as described in Sardana et al. (1996, Plant Cell Reports 15: 677-681). In this method, the codon utilization standard deviation, a measure of the codon utilization trend, can be calculated by first finding the square of the proportional deviation of utilization of each codon of the native gene in relation to that of highly expressed plant genes, followed by a calculation of the square of the mean deviation. The formula used is: 1 SDCU = n = 1 N [(Xn -Yn) / Yn] 2 / N, where Xn refers to the frequency of use of codon n in highly expressed plant genes, where Yn refers to frequency of use of codon n in the gene and interest and N refers to the total number of codons in the gene of interest. A codon utilization table for highly expressed dicot genes is compiled using data from Murray et al. (1989, Nuc AcID's Res. 17: 477-498).

[00309] A method for optimizing the nucleic acid sequence according to the preferred use of the codon for a particular type of plant cell is based on the direct use, without performing any extra statistical calculations, of codon optimization tables such as those available online at the Codon Utilization Database through the DNA bank of NIAS (National Institute of Agrobiological Sciences 1 National Institute of Agrobiological Sciences) in Japan (http: // www (dot) kazusa (dot) or (dot) jp / codon /). The Codon Utilization Database contains codon utilization tables for several different species, with each codon utilization table having been statistically determined based on data from Genbank.

[00310] Using the Tables above to determine the most preferred or most favored codons for each amino acid in a particular species (eg, rice), a naturally occurring nucleotide sequence encoding a protein of interest to have the codon optimized for that particular plant species. This is done by replacing codons that may have a low statistical incidence in the genome of the particular species with corresponding codons, in relation to an amino acid, that are statistically more favored. However, one or more less favored codons can be selected to exclude existing restriction sites, to create new ones in potentially useful unions (5 'and 3' terminals to add the signal peptide or termination cassettes, internal sites that can be used for cut and assemble segments to produce a correct full-length sequence), or to eliminate nucleotide sequences that may negatively affect stability or mRNA expression.

[00311] The naturally occurring coding nucleotide sequence may, before any modification, already contain a number of codons that correspond to a statistically favored codon in a particular plant species. Therefore, codon optimization of the native nucleotide sequence may comprise determining which codons, within the native nucleotide sequence, are not statistically favored with respect to a particular plant and modifying those codons according to a codon usage table for the plant particular to produce an optimized codon derivative. A modified nucleotide sequence can be totally or partially optimized for using the plant's codon as long as the protein encoded by the modified nucleotide sequence is produced at a higher level than the protein encoded by the corresponding naturally occurring or native gene. The structure of synthetic genes altering the use of the codon is described, eg, in PCT Patent Application (Patent Cooperation Treaty 1 Patent Cooperation Treaty) 93/07278.

[00312] According to some applications of the present patent application, the exogenous polynucleotide is a non-coding RNA.

[00313] As used herein, the term "non-coding RNA" refers to an RNA molecule that does not encode an amino acid sequence (a polypeptide). Examples of such non-coding RNA molecules include, but are not limited to, an antisense RNA, a premiRNA (precursor to a microRNA) or a precursor to an RNA that interacts with Piwi (piRNA).

[00314] Non-limiting examples of non-coding RNA polynucleotides are given in SEQ IDs. No. 215, 257, 258 and 469.

[00315] Thus, the present invention application covers the nucleic acid sequences described above, fragments of them, hybridizing sequences found in them, homologous sequences thereof, sequences encoding similar polypeptides with different uses of codons, altered sequences characterized by mutations, as exclusion, introduction or substitution of one or more nucleotides, occurring naturally or induced by man, either randomly or in the objectified way.

[00316] The present application provides an isolated polynucleotide, comprising a nucleic acid sequence of at least about 80%, at least about 81%, at least about 82%, at least about from 83% at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89 at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, eg 100% identical to the polynucleotide selected from the group consisting of SEQ IDs. No. 1- 469, 763-3704 and 3705.

[00317] According to some applications of the present patent application, the nucleic acid sequence is capable of increasing the efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, growth rate, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress in a plant.

[00318] According to some applications of the present application, the isolated polynucleotide comprising a nucleic acid sequence is selected from the group consisting of SEQ IDs. No. 1-469, 763-3704 and 3705.

[00319] According to some applications of the present patent application, the isolated polynucleotide is established by SEQ IDs. No. 1469, 763-3704 or 3705.

[00320] The present application provides an isolated polynucleotide, comprising a nucleic acid sequence encoding a polypeptide comprising an amino acid sequence of at least about 80%, at least about 81%, at least about at least 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88 at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or, say, 100% homologous to the amino acid sequence selected from the group consisting of SEQ IDs. No. 470-762, 3706-5858, 5860-5910, 5912, 5914-5923, 59256046 and 6047.

[00321] According to some applications of the present patent application, the amino acid sequence is capable of increasing the efficiency in the use of fertilizer (eg, efficiency in the use of nitrogen), oil content, production, rate growth, biomass, vigor, fiber production, fiber quality and / or tolerance to abiotic stress in a plant.

[00322] The present application provides an isolated polynucleotide, comprising a nucleic acid sequence encoding a polypeptide comprising the amino acid sequence selected from the group consisting of SEQ IDs. No. 470-762, 3706-5858, 5860-5910, 5912, 25 5914-5923, 5925-6046 and 6047.

[00323] In accordance with an aspect of some applications of the present patent application, a nucleic acid structure is provided, comprising the polynucleotide isolated from the present patent application and a promoter for directing the transcription of the nucleic acid sequence in a host cell.

[00324] The present application provides an isolated polypeptide, comprising an amino acid sequence of at least about 80%, at least about 81%, at least about 82%, at least about 83 at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 93%, at least about 94%, at least , about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or, say, 100% homologous to an amino acid sequence selected from the group consisting of SEQ IDs. No. 470-762, 3706-5858, 5860-5910, 5912, 5914-5923, 5925-5 6046 and 6047.

[00325] According to some applications of the present application, the polypeptide comprising an amino acid sequence is selected from the group consisting of SEQ IDs. No. 470-762, 3706-5858, 5860-5910, 5912, 5914-5923, 5925-6046, 6047, 5859, 5911, 5913 and 5924.

[00326] According to some applications of the present patent application, the polypeptide is established by SEQ IDs. No. 470-762, 37065858, 5860-5910, 5912, 5914-5923, 5925-6046, 6047, 5859, 5911, 5913 and 5924.

[00327] The present patent application also covers fragments of the polypeptides described above and of polypeptides showing mutations, such as exclusions, introductions or substitutions of one or more amino acids, occurring naturally or induced by man, either randomly or in a targeted manner.

[00328] The term "plant", as used here, encompasses whole plants, ancestors and progenies of plants and parts of the plant, including seeds, buds, stems, roots (including tubers) and cells, tissues and organs of the plant. The plant can be in any form, including suspension cultures, embryos, meristematic regions, callous tissue, leaves, gametophytes, sporophytes, pollen and microspores. Plants that are particularly useful in the methods of the present patent application include all plants that belong to the superfamily Viridiplantae, in particular monocots and dicots, including a forage or forage legume, ornamental plant, food crop, tree or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp. , Brassica spp., Bruguiera gymnorrhiza, African burkea, Leafy loofah, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chacoomeles spp., Cinnamomum cassia, Coffea arabica, Colophosumum, Colophosumum Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp. , Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Dibeteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehraffia spac. , Eucalypfus spp., Euclea schimperi, Eulalia vi / losa, Pagopyrum spp., Feijoa sellowlana, Fragaria spp., Flemingia spp, Freycinetia banksli, Geranium thunbergii, GinAgo biloba, Glycine javanica, Gliricidia spp. coleosperm, Hedysarum spp., Hemaffhia altissima, Heteropogon contoffus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hypeffhelia dissolute, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp. , Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago saliva, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Ornithopus s pp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp.,
Picea glauca, Pinus spp., Pisum sativam, Podocarpus totara, Pogonarthria fleckii, Pogonaffhria squarrosa, Populus spp., Prosopis cineraria, Pseupontosuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus sppisis Risphis, Rhaphisis, Rhaphis , Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp.,
Salix spp., Schyzachyrium sanguineum, Sciadopitys vefficillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Trmed spic. heterophylla, Vaccinium spp., Vicia spp., Vitis vinífera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, kale, canola, carrots, cauliflower, celery, cauliflower, celery flax, kale, lentil, rapeseed, okra, onion, potato, rice, soy, straw, beet, sugar cane, sunflower, tomato, pumpkin tea, corn, wheat, barley, rye, oats, peanuts, peas, lentils and alfalfa, cotton, rapeseed, canola, pepper, sunflower, tobacco, eggplant, eucalyptus, a tree, an ornamental plant, a perennial grass and a forage crop. Alternatively, algae and other non-Viridiplantae can be used for the methods of the present application.

[00329] According to some applications of the present patent application, the plant used by the method of the present patent application is a crop plant such as rice, corn, wheat, barley, peanut, potato, sesame, olive , palm oil, banana, soy, sunflower, canola, sugar cane, alfalfa, millet, legumes (beans, peas), flax, lupine, rapeseed, tobacco, poplar and cotton.

[00330] According to some applications of the present patent application, the plant is a dicotyledonous plant.

[00331] According to some applications of the present patent application, the plant is a monocotyledonous plant.

[00332] According to some applications of the present patent application, a plant cell is presented exogenously expressing the polynucleotide of some applications of the present patent application, the nucleic acid structure of some applications of the present application patent and / or the polypeptide of some applications of the present application.

[00333] According to some applications of the present patent application, the expression of the exogenous polynucleotide of the present patent application within the plant is affected by the transformation of one or more cells of the plant with the exogenous polynucleotide, followed by generation of a mature plant from the transformed cells and cultivating the mature plant under suitable conditions to express the exogenous polynucleotide within the mature plant.

[00334] According to some applications of the present patent application, the transformation is effected by the introduction of a nucleic acid structure in the plant cell that includes the
exogenous polynucleotide from some applications of the present application and at least one promoter to guide the transcription of the exogenous polynucleotide in a host cell (a plant cell).
Additional details of suitable transformation approaches are provided below.

[00335] As mentioned, the nucleic acid structure, according to some applications of the present patent application, comprises a promoter sequence and the polynucleotide isolated from the present patent application.

[00336] According to some applications of the present patent application, the isolated polynucleotide is operably linked to the promoter sequence.

[00337] A coding nucleic acid sequence is "operationally linked" to a regulatory sequence (e.g., the promoter) if the regulatory sequence is able to exert a regulatory effect on the coding sequence attached to it.

[00338] As used here, the term "promoter" refers to a region of DNA that lies upstream of the transcriptional initial site of a gene to which RNA polymerase binds to initiate RNA transcription. The promoter controls where (eg, which portion of a plant) and / or when (eg, at what stage or condition in an organism's lifetime) the gene is expressed.

[00339] According to some applications of the present patent application, the promoter is heterologous to the isolated polynucleotide and / or the host cell.

[00340] As used herein, the term "heterologous promoter" refers to a promoter of a different species or the same species, but of a different gene locus, from the isolated plinucleotide sequence.

Any suitable promoter sequence can be used by the nucleic acid structure of the present application. Preferably, the promoter is a constitutive promoter, a tissue-specific or inducible to abiotic stress promoter.

[00342] According to some applications of the present patent application, the promoter is a plant promoter, which is suitable for expression of the exogenous polynucleotide in a plant cell.

Suitable constitutive promoters include, for example, the CaMV 35S promoter [SEQ ID. No. 6048 (pQFNC); SEQ ID. No. 6049 (PJJ 35S for Brachypodium); SEQ ID NO. 6050 (Odell et al., Nature 313: 810-812, 1985)], Arabidopsis At6669 promoter (SEQ ID No. 6051; see PCT Publication No. W004081173A2 or the new At6669 promoter (SEQ ID No. 6052); corn Ubi 1 (polyubiquitin corn 1, SEQ ID No. 6053; Christensen et al., Plant Sol. Biol. 18: 675-689, 1992; Taylor et al., Plant Cell Rep 12: 491- 495, 1993); 1 of rice (SEQ ID No. 6054, McElroy et al., Plant Cell 2: 163-171, 1990); pEMU (Last et al., Theor. Appl. Genet. 81: 581-588, 1991); CaMV 19S (Nilsson et al., Physiol. Plant 100: 456-462, 1997); GOS2 (SEQ ID No. 6055, by Pater et al, Plant J Nov; 2 (6): 837-44, 1992); Ubi promoter 1 (SEQ ID. No. 6056); RBCS promoter (SEQ ID No.: 6057); Rice cyclophylline (Bucholz et al, Plant Mol Biol. 25 (5): 837-43, 1994); corn histone H3 ( Lepetit et al, Mol. Gen. Genet. 231: 276-285, 1992); Actin 2 (An et al, Plant J. 10 (1); 107-121, 1996) and Super MAS Synthetic (Ni et al., The Plant Journal 7: 661-76, 1995) .Other constitutive promoters are included in Patent Nos. Inter-American No. 5,659,026, 5,608,149; 5,608,144; 5,604,121; 5,569,597: 5,466,785; 5,399,680; 5,268,463; and 5,608,142.

[00344] Suitable tissue specific promoters include, but are not limited to, leaf specific promoters [eg, AT5G06690 (Thioredoxin) (high expression, SEQ ID N ° 6058), AT5G61520 (AtSTP3) (low expression, ID SEQ No. 6059) described in Buttner et al 2000 Plant, Cell and Environment 23, 175-184, or the promoters described in Yamamoto et al., Plant J. 12: 255-265, 1997; Kwon et al., Plant Physiol. 105: 357-67, 1994; Yamamoto et al., Plant Cell Physiol. 35: 773-778, 1994; Gotor et al., Plant J. 3: 509-18, 1993; Orozco et al., Plant Mol. Biol. 23: 1129-1138, 1993; and Matsuoka et al., Proc. Natl. Acad. Sci. USA 90: 9586-9590, 1993; as well as Arabidopsis STP3 (AT5G61520) promoter (Buttner et al., Plant, Cell and Environment 23: 175-184, 2000)], preferred seed promoters [eg, Napin (originated from Brassica napus and characterized by a specific seed promoting activity; Stuitje AR et al. Plant Biotechnology Journal 1 (4): 301-309; SEQ ID No. 6060 from specific seed genes (Simon, et al., Plant Mol. Biol. 5 . 191, 1985; Scofield, et al., J. Biol. Chem. 262: 12202, 1987; Baszczynski, et al., Plant Moi. Biol. 14: 633, 1990), PG5a rice (US 7,700,835), early Arabidopsis BAN seed development (SEQ ID No. 6061, US 2009/0031450 Al), late Arabidopsis ABI3 seed development (SEQ ID No. 6062) (Ng et al., Plant Molecular Biology 54: 25-38, 2004), Brazil nut albumin (Ervilharson 'et al., Plant Mol. Biol. 18: 235-245, 1992), legumes (Eliis, et al. Plant Mol. Biol. 10: 203214, 1988), Glutelin ( rice) (Takaiwa, et al., Mol. Gen. Genet. 208: 15-22, 1986; T akaiwa, et al., FEBS Letts. 221: 43-47, 1987), Zein (Matzke et al Plant Mol Biol, 143): 32332 1990), napA (Stalberg, et al, Plant 199: 515-519, 1996), wheat SPA (Albanietal, Plant Cell , 9: 171- 184, 1997), sunflower oleosin (Cummins, et al., Plant Mol. Biol. 19: 873-76, 1992)], specific endosperm promoters [eg, LMW and HMW wheat, glutenin-1 (Thomas and Flavell, The Plant Cell 2: 1171-1180, 1990; Mol Gen Genet 216: 81-90, 1989; NAR 17: 461-2), wheat a, beg gliadins (EMBO3: 1409-15, 1984), barley promoter ltrl, barley Bl, C, D hordein (Theor Appl Gen 98: 1253-62, 1999; Plant J 4: 343-55, 1993; Mol Gen Genet 250: 750-60, 1996), Barley DOF (hena et al, The Plant Journal, 116 (1): 53- 62, 1998), Biz2 (EP99106056.7), Barley SS2 (Guerin and Carbonero Plant Physiology 114: 1 55-62, 1997), Wheat Tarp60 ( Kovalchuk et al., Plant Mol Biol 71: 81-98, 2009), Barley D-hordein (DHor) and B-hordein (B-Hor) (Agnelo Furtado, Robert J. Henry and Alessandro Pellegrineschi (2009)], promoter Synthetic (Vicente-Carbajosa et al., Plant J. 13: 629-640, 1998), NRP33 rice prolamine, Glb-1 rice globulin (Wu et al, Plant Cell Physiology 39 (8) 885-889, 1998), REB rice alphaglobulin rice / OHP-1 (Nakase et al. Plant Mol. Biol. 33: 513-S22, 1997), rice rice ADP-glucose PP (Trans Res 6: 157- 68, 1997), ESR maize gene family (Plant J 12: 235-46, 1997), sorghum gamakafirine (PMB 32: 1029-35, 1996)], embryonic specific promoters [eg, OSH1 rice (Sato et al, Proc. Natl. Acad. Sci. USA, 93: 81178122), KNOX (Postma-Haarsma et al, Plant Mol. Biol. 39: 25771, 1999), oleosin rice (Wu et at, J. Biochem, 123: 386, 1998)] and specific flower promoters [eg, AtPRP4, chalene synthase (chsA) (Van der Meer , et al., Plant Mol. Biol. 15, 95-109, 1990), LAT52 (Twell et al Mol. Gen Genet. 217: 240- 245; 1989), Arabidopsis apetala-3 (Tilly et al., Development 125 : 1647- 57, 1998), Arabidopsis APETALA 1 (AT1G69120, AP1) (SEQ ID NO: 6063) (Hempel et al., Development 124: 3845-3853, 1997)] and root promoters [e.g. the ROOTP promoter [SEQ ID. No. 6064]; promoters of ExpB5 rice and ExpBl barley (Won et al. Mol. Cells 30: 369-376, 2010); Arabidopsis monoterpene synthase promoter (AT3G25820) (Chen et al., Plant Phys 135: 1956-66, 2004); Arabidopsis Phol promoter (SEQ ID NO: 6065, Hamburger et al., Plant Cell. 14: 889-902, 2002), which is also slightly stress-induced Pi].

[00345] Promoters inducible by abiotic stress include, but are not limited to, salt inducible promoters such as RD29A (YamaguchiShinozalei et al., Mol. Gen. Genet. 236: 331-340, 1993); drought-inducible promoters such as the rabl7 promoter gene (Pla et. al., Plant Mol. Biol. 21: 259-266, 1993), the corn rab28 promoter gene (Busk et. al., Plant J. 11: 1285- 1295, 1997 and the corn Ivr2 promoter gene (Pelleschi et. Al., Plant Mol. Biol. 39: 373-380, 1999); heat-inducible promoters such as the tomato hsp80 promoter (U.S. Patent No. 5,187. 267).

The nucleic acid structure of some applications of the present application may further include an appropriate selectable marker and / or an origin of replication. According to some applications of the present patent application, the nucleic acid structure used is a bridge vector, which can propagate both in E. coli (where the structure comprises an appropriate selectable marker and the origin of replication) and can be compatible with cell propagation. The structure according to the present patent application can be, for example, a plasmid, a bacmid, a phagemid, a cosmid, a phage, a virus or an artificial chromosome.

[00347] The nucleic acid structure of some applications of the present patent application can be used to transform plant cells in a stable or transient manner. In stable transformation, the exogenous polynucleotide is integrated into the plant's genome and, as such, represents a stable and inherited trait. In transient transformation, the exogenous polynucleotide is expressed by the transformed cell, but is not integrated into the genome and, as such, represents a transient trait.

[00348] There are several methods for introducing foreign genes, both in monocots and dicots (Potrykus, I., Annu. Rev. Plant. Physiol, Plant. Mol. Biol. (1991) 42: 205225; Shimamoto et al., Nature (1989) 338: 274-276).

[00349] The principle-of-cause methods of stable integration of exogenous DNA into plant genomic DNA include two main approaches:
Agrobacterium-mediated gene transfer: Klee et al. (1987) Annu. Rev. Plant Physiol. 38: 467-486; Klee and Rogers in Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes, eds. Schell, J. and Vasil, LK, Academic Publishers, San Diego, Calif. (1989) p. 2-25; Gatenby, in Plant Biotechnology, eds. Kung, S. and Arntzen, CJ, Butterworth Publishers, Boston, Mass. (1989) p. 93-112.

[00350] Direct DNA absorption: Paszkowski et al., In Cell Culture and Somatic Cell Genetics of Plants, Vol. 6, Molecular Biology of Plant Nuclear Genes eds. Schell, J. and Vasil, L. K., Academic Publishers, San Diego, Calif. (1989) p. 52-68; including methods for direct DNA absorption in protoplasts, Toriyama, K. et al. (1988) Bio / Technology 6: 1072-1074. DNA absorption induced by the brief electrical shock of plant cells: Zhang et al. Plant Cell Rep. (1988) 7: 379384. Fromm et al. Nature (1986) 319: 791-793. Injection of DNA into plant cells or tissues by particle bombardment, Klein et al. Bio / Technology (1988) 6: 559-56; McCabe et al. Bio / Technology (1988) 6: 923-926; Sanford, Physiol. Plant. (1990) 79: 206-209; for the use of micropipette systems: Neuhaus et al., Theor. Appl. Genet. (1987) 75: 30-36; Neuhaus and Spangenberg, Physiol. Plant. (1990) 79: 213-217; transformation of particles of glass fibers or silicon carbide from cell cultures, embryos or callous tissue, US Patent No. 5,464,765 or by direct DNA incubation with germinating pollen, DeWet et al. in Experimental Manipulation of Ovule Tissue, eds. Chapman, G. P. and Mantell, S. H. and Daniels, W. Longman, London, (1985) p. 197-209; and Ohta, Proc. Natl. Acad. Sci. USA (1986) 83: 715-719.

[00351] The Agrobacterium system includes the use of plasmid vectors that contain defined segments of DNA that integrate into the plant's genomic DNA. Inoculation methods for plant tissue vary depending on the species of the plant and the delivery system of the Agrobacterium. A widely used approach is the leaf disc procedure that can be performed with any tissue explant that offers a good source for beginning to differentiate the entire plant. See, eg, Horsch et al. in Plant Molecular Biology Manual A5, Kluwer Academic Publishers, Dordrecht (1988) p. 1-9. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicots.

[00352] There are several methods of direct DNA transfer to plant cells. In electroporation, protoplasts are briefly exposed to a strong electric field. In microinjection, DNA is mechanically injected directly into cells using very small micropipettes. In the bombardment of microparticles, DNA is absorbed in microprojectiles such as magnesium sulfate crystals or tungsten particles and microprojectiles are physically accelerated in plant cells or tissues.

[00353] After the stable transformation, plant propagation is carried out. The most common method of plant propagation is by seed. Regeneration by seed propagation, however, has the deficiency that due to heterozygosity there is a lack of uniformity in the crop, since the seeds are produced by plants according to the genetic variances governed by Mendelian norms. Basically, each seed is genetically different and each will grow with its own specific traits. Therefore, it is preferable that the transformed plant is produced in such a way that the regenerated plant has identical traits and characteristics as the transgenic matrix plant. Therefore, it is preferable that the transformed plant is regenerated by micropropagation that provides rapid, consistent reproduction of the transformed plants.

[00354] Micropropagation is a process of growing new generation plants from a piece of tissue that has been removed from a selected parent plant or cultivar. This process allows the mass reproduction of plants that have the preferred tissue expressing the fusion protein. The new generation plants that are produced are genetically identical to the original plant and have all their characteristics. Micropropagation allows the mass production of quality plant material in a short period of time and offers a rapid multiplication of cultivars selected to preserve the characteristics of the original transgenic or transformed plant. The advantages of plant cloning are the speed of multiplication of the plant and the quality and uniformity of the plants produced.

[00355] Micropropagation is a multi-stage procedure that requires changing the culture medium or culture conditions between stages. Thus, the micropropagation process involves four basic stages: Stage one, initial tissue culture; stage two, multiplication of tissue culture; stage three, differentiation and formation of the plant; and fourth stage, culture and greenhouse strengthening. During stage one, the initial tissue culture, the tissue culture is established and certified as being free of contaminants. During stage two, the initial tissue culture is multiplied until a sufficient number of tissue samples are produced to meet production objectives. During stage three, the tissue samples grown in stage two are divided and grown on individual seedlings. In stage four, the transformed seedlings are transferred to a greenhouse for strengthening, where the plants' tolerance to light is gradually increased so that it can be grown in the natural environment.

[00356] According to some applications of the present patent application, transgenic plants are generated by the transient transformation of leaf cells, meristematic cells or the entire plant.

[00357] Transient transformation can be carried out by any of the direct DNA transfer methods described above or by viral infection using modified plant viruses.

[00358] Viruses that have been shown to be useful for the transformation of plant hosts include CaMV, the tobacco mosaic virus (TMV I tobacco mosaic virus), bromine mosaic virus (BMV I brome mosaic virus) and the Common Bean Mosaic Virus (BV or BCMV I common mosaic virus). Plant transformation using plant viruses is described in U.S. Patent No. 4,855,237 (golden mosaic bean virus; BGV), EP-A 67,553 (TMV), Japanese Application Published No. 63-14693 (TMV), EPA 194,809 (BV), EPA 278,667 (BV); and Gluzman, Y. et al., Communications in Molecular Biology: Viral Vectors, Cold Spring Harbor Laboratory, New York, pp. 172-189 (1988). Pseudoviral particles for use in the expression of foreign DNA in many hosts, including plants, are described in WO 87/06261.

[00359] According to some applications of the present patent application, the virus used for transient transformation is avirulent and is therefore unable to cause severe symptoms such as reduced growth rate, mosaic, ring stains, leaf curl , yellowing, stretch marks, vesicle formation, tumor formation and corrosion. A suitable avirulent virus can be a naturally occurring avirulent virus or an artificially attenuated virus. The attenuation of the virus can be carried out using methods well known in the art, including, but not limited to, sublethal heating, chemical treatment or by mutagenesis techniques directed as described, eg, by Kurihara and Watanabe (Molecular Plant Pathology 4: 259-269, 2003), Galon et al. (1992), Atreya et al. (1992) and Huet et al. (1994).

[00360] Suitable viral strains can be obtained from available sources, such as, for example, from the American Type Culture Collection (ATCC 1 American Type Culture Collection) or by isolating infected plants. Isolation of viruses from infected plant tissues can be accomplished by techniques well known in the art, as described, e.g., by Foster and Tatlor, Eds. "Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998. It is believed that quickly, the tissues of an infected plant contain a high concentration of a virus suitable, preferably young leaves and flower petals, are crushed in a buffer solution (eg, phosphate buffer solution) to produce a virus-infected sap that can be used in subsequent inoculations.

[00361] The structure of plant RNA viruses for the introduction and expression of exogenous non-viral polynucleotide sequences in plants is demonstrated by the references above, as well as by Dawson, WO et al., Virology (1989) 172: 285- 292; Takamatsu et al. EMBO J. (1987) 6: 307-311; French et al. Science (1986) 231: 1294-1297; Takamatsu et al. FEBS Letters (1990) 269: 7376; and U.S. Patent No. 5,316,931.

[00362] When the virus is a DNA virus, appropriate modifications can be made to the virus itself. Alternatively, the virus can first be cloned into a bacterial plasmid to facilitate the structure of the desired viral vector with the foreign DNA. Then, the virus can be extracted from the plasmid. If the virus is a DNA virus, a bacterial origin of replication can be attached to the viral DNA, which is then replicated by the bacteria. The transcription and translation of this DNA will produce the coat protein that will encapsulate viral DNA. If the virus is an RNA virus, the virus is usually cloned as a cDNA and introduced into a plasmid. Then, the plasmid is used to make all the structures. Then, the RNA virus is produced by transcribing the plasmid's viral sequence and translating the viral genes to produce the coating protein (s) that encapsulate the viral RNA.

[00363] In one application, a viral polynucleotide from a plant is provided, in which the native coating protein encoding the sequence has been excluded from a viral polynucleotide, a non-native viral coating protein from the plant encoding the sequence and a non-promoter native, preferably the subgenomic promoter of the non-native coating protein encoding the sequence, capable of expressing itself in the plant host, in the wrapper of the plant's recombinant viral polynucleotide and guaranteeing a systemic infection of the host by the plant's recombinant viral polynucleotide, was introduced . Alternatively, the coat protein gene can be inactivated by introducing the non-native polynucleotide sequence within it, so that a protein is produced. The plant's recombinant viral polynucleotide may contain one or more additional non-native subgenomic promoters. Each non-native subgenomic promoter is capable of transcribing or expressing adjacent genes or polynucleotide sequences in the plant host and unable to recombine with one another and with the native subgenomic promoters. Non-native (foreign) polynucleotide sequences can be introduced adjacent to the native viral subgenomic promoter of the plant or the native viral subgenomic promoter and the non-native viral subgenomic promoter of the plant, if there is more than one polynucleotide sequence is included. The non-native polynucleotide sequences are transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the desired products.

[00364] In a second application, a recombinant viral polynucleotide from the plant is provided, as in the first application, except that the native coat protein encoding the sequence is placed adjacent to one of the genomic promoters of the non-native coat protein instead of a coding sequence for the non-native coating protein.

[00365] In a third application, a recombinant plant viral polynucleotide is provided, in which the native coat protein gene is adjacent to its subgenomic promoter and one or more non-native subgenomic promoters has been (ram) inserted into the polynucleotide viral. The introduced non-native subgenomic promoters are capable of transcribing or expressing adjacent genes in a plant host and are unable to recombine with one another and with native subgenomic promoters. Non-native polynucleotide sequences can be introduced adjacent to the plant's non-native viral subgenomic promoters so that the sequences are transcribed or expressed in the host plant under the control of the subgenomic promoter to produce the desired product.

[00366] In a fourth application, a recombinant viral polynucleotide from the plant is provided, as in the third application, so that the coding sequence for the native coating protein is replaced by a coding sequence for the non-native coating protein.

[00367] Viral vectors are encapsulated by the coat proteins encoded by the plant's recombinant viral polynucleotide to produce a recombinant plant virus. The recombinant plant viral polynucleotide or the recombinant plant virus is used to infect appropriate host plants. The plant's recombinant viral polynucleotide is capable of replication in the host, systemic diffusion in the host, and transcription or expression of foreign gene (s) (exogenous polynucleotide) in the host to produce the desired protein.

[00368] Techniques for inoculating viruses to plants can be found in Foster and Taylor, eds. "Plant Virology Protocols: From Virus Isolation to Transgenic Resistance (Methods in Molecular Biology (Humana Pr), Vol 81)", Humana Press, 1998; Maramorosh and Koprowski, eds. "Methods in Virology" 7 vols, Academic Press, New York 1967-1984; Hill, S.A. "Methods in Plant Virology", Blackwell, Oxford, 1984; Walkey, D.G.A. "Applied Plant Virology", Wiley, New York, 1985; and Kado and Agrawa, eds. "Principies and Techniques in Plant Virology", Van Nostrand-Reinhold, New York.

[00369] In addition to the above, the polynucleotide of the present patent application can also be introduced into a chloroplast genome, thus allowing the expression of chloroplast.

[00370] A technique for introducing exogenous polynucleotide sequences into the chloroplast genome is known. This technique involves the following procedures. First, plant cells are chemically treated in order to reduce the number of chloroplasts per cell to approximately one. Then, the exogenous polynucleotide is introduced by bombarding particles in the cells with the aim of introducing at least one molecule of the exogenous polynucleotide into the chloroplasts. The exogenous polynucleotides are selected so that they can be integrated into the chloroplast genome through homologous recombination that is promptly carried out by the enzymes inherent to the chloroplast. To that end, the exogenous polynucleotide includes, in addition to a gene of interest, at least one extension of the polynucleotide that is derived from the chloroplast genome. In addition, the exogenous polynucleotide includes a selectable marker that serves, by sequential selection procedures, to verify whether all or substantially all copies of the chloroplast genomes, after this selection, will include the exogenous polynucleotide. Additional details related to this technique are found in North American Patents No. 4,945,050 and 5,693,507 which are incorporated herein by reference. Thus, a polypeptide can be produced by the chloroplast protein expression system and integrates with the internal membrane of the chloroplast.

[00371] Since the processes that increase the efficiency in the use of nitrogen, efficiency in the use of fertilizer, oil content, production, seed production, fiber production, fiber quality, fiber length, growth rate, biomass , vigor and / or tolerance to abiotic stress in a plant may involve multiple genes that act additively or in synergy (see, eg, in Quesda et al., Planta Physiol. 130: 951-063, 2002), the present The invention patent application also provides for the expression of a plurality of exogenous polynucleotides in a single host plant, thereby achieving a superior effect on oil content, production, growth rate, biomass, vigor and / or tolerance to abiotic stress. a plant.

[00372] The expression of a plurality of exogenous polynucleotides in a single host plant can be carried out by co-introducing multiple nucleic acid structures, each including a different exogenous polynucleotide, in a single plant cell. The transformed cell can then be regenerated into a mature plant using the methods described above.

Alternatively, the expression of a plurality of exogenous polynucleotides in a single host plant can be carried out by co-introducing, in a single cell of the plant, a single nucleic acid structure including a plurality of different exogenous polynucleotides. This structure can be designed with a single promoter sequence that can transcribe a polycistronic messenger RNA including all different sequences of exogenous polynucleotide. In order to allow the co-translation of the different polypeptides encoded by the polycistronic messenger RNA, the polynucleotide sequences can be interconnected through a sequence of the internal ribosome entry site (IRES I internal ribosome entry site) that facilitates the translation of the positioned polynucleotide sequences downstream of the IRES sequence. In this case, a transcribed polycistronic RNA molecule that encodes the different polypeptides described above will be translated both at the 5 'end that has the cap and in the two internal IRES sequences of the polycistronic RNA molecule to thus produce all the different polypeptides in the cell . Alternatively, the structure can include several promoter sequences, each linked to a different exogenous polynucleotide sequence.

[00374] The plant cell transformed with the structure, including a plurality of different exogenous polynucleotides, can be regenerated in a mature plant using the methods described above.

Alternatively, the expression of a plurality of exogenous polynucleotides in a single host plant can be effected by introducing different nucleic acid structures, including different exogenous polynucleotides, in a plurality of plants. The regenerated transformed plants can then be crossed and the resulting progeny selected to obtain superior traits of tolerance to abiotic stress, efficiency in the use of water, efficiency in the use of fertilizer, growth, biomass, production and / or vigor, using conventional techniques of plant breeding.

[00376] According to some applications of the present patent application, the method further comprises the cultivation of the plant expressing the exogenous polynucleotide under abiotic stress.

[00377] Non-limiting examples of abiotic stress conditions include salinity, osmotic stress, drought, water deprivation, excess water (eg, flooding, waterlogging), etiolation, low temperature (eg, cold stress ), high temperature, heavy metal toxicity, anaerobiosis, nutrient deficiency, excess nutrients, atmospheric pollution and UV irradiation.

[00378] According to some applications of the present patent application, the method further comprising plant growth expressing the exogenous polynucleotide under fertilizer limiting conditions (e.g., nitrogen limiting conditions). Non-limiting examples include plant growth in soils with low nitrogen content (40-50% nitrogen content present under normal and ideal conditions), or even in severe nitrogen deficiency (0 to 10% nitrogen content present under normal and ideal conditions).

[00379] Accordingly, the present patent application covers plants that exogenously express the polynucleotide (s), nucleic acid structures and / or the polypeptide (s) of the present patent application of invention.

[00380] Once expressed within the plant cell or the entire plant, the level of the polypeptide encoded by the exogenous polynucleotide can be determined by methods well known in the art, such as activity assays, Western blots using antibodies capable of specifically binding to the polypeptide, Enzyme-Linked Immunoabsorbent Assay (ELISA I enzymelinked immuno sorbent assay), radioimmunoassays (RIA 1 radio-immuno-assays), immunohistochemistry, immunocytochemistry, immunofluorescence and other similar methods.

[00381] Methods for determining the level of the transcribed RNA of the exogenous polynucleotide in the plant are well known in the art and include, for example, Northern blot analysis, polymerase chain reaction analysis with reverse transcriptase (RT-PCR 1 reverse transcription polymerase chain reaction) (including quantitative, semi-quantitative or real-time RT-PCR) and in situ hybridization to RNA.

[00382] The information and notes of the sequence not covered by the present teachings can be used in favor of the classic improvement. Thus, the subsequence data of those polynucleotides described above can be used as markers for marker assisted selection (MAS 1 marker assisted selection), in which a marker is used for indirect selection of a determinant or genetic determinants (s) of a trait of interest (eg, biomass, growth rate, oil content, production, tolerance to abiotic stress, water use efficiency, nitrogen use efficiency and / or fertilizer use efficiency). The nucleic acid data of the present teachings (DNA or RNA sequence) may contain or be linked to polymorphic sites or genetic markers in the genome such as the restriction fragment length polymorphism (RFLP 1 restriction fragment length polymorphism), microsatellites and polymorphism single nucleotide polymorphism), genetic imprinting (DFP 1 DNA fingerprinting), amplified fragment length polymorphism (AFLP 1 amplified fragment length polymorphism), expression level polymorphism, encoded polypeptide polymorphism and any other polymorphism in DNA or RNA sequence.

[00383] Examples of assisted marker selections include, but are not limited to, the selection of a morphological trait (eg, a gene that affects shape, color, male sterility or resistance such as the presence or absence of edge, leaf sheath color, height, grain color, rice aroma); selection of a biochemical trait (eg, a gene that encodes a protein that can be extracted and observed; eg, isozymes and reserve proteins); selection of a biological trait (eg, (pathogenic breeds or insect biotypes based on the host pathogen or interaction with the host parasite) can be used as a marker as long as the genetic makeup of an organism can affect its susceptibility to pathogens or parasites).

[00384] The polynucleotides and polypeptides described above can be used in a wide variety of economic plants, safely and cost-effectively.

[00385] Vegetable strains that exogenously express the polynucleotide or polypeptide of the present patent application are screened to identify those that show the greatest increase in the desired characteristic of the plant.

[00386] Therefore, according to a further application of the present application for a patent, a method of evaluating a characteristic of a plant is provided, the method comprising: (a) expressing in a plant or in a respective part the structure nucleic acid from some applications of the present application (described above); and (b) to evaluate a characteristic of a plant in comparison with a wild type plant of the same type (eg, the same species, eg, having the same genotype, except if not transformed with the claimed biomolecules); thus evaluating the characteristic of the plant.

[00387] In accordance with an aspect of some applications of the present patent application, a method is provided for growing a crop, comprising sowing seeds and / or planting seedlings of a plant transformed with the present polynucleotides patent application, for example, the polynucleotides encoding the polypeptide for some applications of the present patent application, characterized in that the plant is derived from selected plants with at least one characteristic selected from the group consisting of increased efficiency in the nitrogen use, increased tolerance to abiotic stress, increased biomass, increased growth rate, increased vigor, increased production and increased production or quality compared to an unprocessed plant.

[00388] According to some applications of the present patent application, the method of cultivating a crop comprises sowing seeds and / or planting seedlings of a plant transformed with an exogenous polynucleotide comprising a nucleic acid sequence that encodes a polypeptide at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85 at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least , about 98%, at least about 99%, e.g., 100% identical to the polypeptide defined in SEQ ID. N ° 470-762, 3706-5858, 5860-5910, 5912, 5914-5923, 5925-6046 or 6047, characterized by the plant being derived from selected plants with at least one characteristic selected from the group consisting of increased efficiency in the nitrogen use, increased tolerance to abiotic stress, increased biomass, increased growth rate, increased vigor, increased production and increased fiber production or quality and increased oil content compared to an unprocessed plant, thus increasing the harvest.

[00389] According to some applications of the present patent application, the method of cultivating a crop comprises sowing seeds and / or planting seedlings of a plant transformed with an exogenous polynucleotide comprising the nucleic acid sequence by at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92% at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% at least about 99%, e.g., 100% identical to the polypeptide defined in SEQ IDs. No. 1-469, 763-3704 or 3705, characterized by the plant being derived from selected plants with at least one characteristic selected from the group consisting of increased efficiency in the use of nitrogen, increased tolerance to abiotic stress, increased biomass , increased growth rate, increased vigor, increased production and increased fiber production or quality and increased oil content compared to an unprocessed plant, thereby increasing the harvest.

[00390] The effect of the transgene (the exogenous polynucleotide that encodes the polypeptide) on tolerance to abiotic stress can be determined using known methods, as detailed below and in the Examples section below.

[00391] Abiotic stress tolerance - Transformed (that is, expressing the transgene) and untransformed (wild type) plants are exposed to an abiotic stress condition, such as water deprivation, suboptimal temperature (low temperature, high temperature), deficiency of nutrients, excess of nutrients, a stress condition caused by salt, osmotic stress, heavy metal toxicity, anaerobiosis, air pollution and UV irradiation.

[00392] Salinity tolerance test - It is expected that transgenic plants with tolerance to high salt concentrations will show better germination, seedling vigor or growth under conditions with high salt levels. Salt stress can be carried out in many ways such as, for example, irrigating plants with a hyperosmotic solution, growing the plants hydroponically in a hyperosmotic culture solution (eg, Hoagland's solution), or subjecting the plants to culture in hyperosmotic growth medium [eg, 50% Murashige-Skoog medium (MS medium)]. As different plants vary considerably in their tolerance to salinity, the concentration of salt in the irrigation water, in the growth solution or in the growth medium can be adjusted according to the specific characteristics of the cultivar or specific variety of the plant, in order to inflict a mild or moderate effect on plant physiology and / or morphology (for guidelines on appropriate concentration, see Bernstein and Kafkafi, Root Growth Under Salinity Stress In: Plant Roots, The Hidden Half 3rd ed. Waisel Y, Eshel A and Kafkafi U. (editors) Marcel Dekker Inc., New York, 2002 and the reference contained therein).

[00393] For example, the salinity tolerance test can be performed by irrigating plants at different stages of development with increasing concentrations of sodium chloride (eg 50 mM, 100 mM, 200 mM, 400 mM NaCl) applied from below and from above to ensure a regular dispersion of the salt. After exposure to the stress condition, the plants are often monitored until the physiological and / or morphological effects appear on the wild type plants. In this way, the external phenotypic appearance, the degree of wilting and the overall success in reaching maturity and the progeny of production are compared between control and transgenic plants.

[00394] The quantitative tolerance parameters measured include, but are not limited to, the average wet and dry weight, the growth rate, the leaf size, the leaf cover (general leaf area), the weight of the seeds generated, the average size of seeds and the number of seeds produced per plant. Transformed plants that do not exhibit substantial physiological and / or morphological effects, or that exhibit greater biomass than wild-type plants, are identified as plants tolerant to abiotic stress.

[00395] Osmotic tolerance test - Osmotic stress tests (including tests with sodium chloride and mannitol) are performed to determine whether an osmotic stress phenotype was specific to sodium chloride or whether it was a phenotype related to general osmotic stress. Plants tolerant to osmotic stress may be more tolerant of drought and / or freezing. For germination experiments under saline and osmotic stress, the medium is supplemented, eg, with 50 mM, 100 mM, 200 mM NaCl or with 100 mM, 200 mM NaCl, 400 mM mannitol.

[00396] Drought tolerance test / Osmotic test - Drought tolerance is performed to identify the genes that give the best survival to the plant after acute water deprivation. To analyze whether transgenic plants are more tolerant to drought, an osmotic stress produced by the non-ionic sorbitol osmolyte in the medium can be performed. Control and transgenic plants are germinated and grown on agar plates on plants for 4 days, after which they are transferred to plates containing 50 mM sorbitol. Treatment causes growth retardation, so both control and transgenic plants are compared, measuring plant weight (wet and dry), yield and measured growth rates such as time to flowering.

[00397] Conversely, dry screens based on soil are made with plants overexpressing the polynucleotides detailed above. Seeds from Arabdopsis control plants, or other transgenic plants overexpressing the polypeptide of the present patent application are germinated and transferred to pots. Drought stress is obtained after irrigation is stopped, accompanied by placing the pots on absorbent paper to improve the rate of soil drying. Transgenic and control plants are compared to each other when most control plants develop severe wilting. The plants receive water again after obtaining a significant fraction of the control plants requiring severe wilting. The plants are classified in comparison with the controls in relation to each of the two criteria: tolerance to drought conditions and recovery (survival) after re-irrigation.

[00398] Cold stress tolerance - To analyze cold stress, mature plants (25 days old) are transferred to chambers at 4 ° C for 1 or 2 weeks, with constitutive light. Subsequently, the plants are returned to the greenhouse. Two weeks later, the damage caused by the cooling period, resulting in growth retardation and other phenotypes, is compared between both control and transgenic plants, measuring the weight of the plant (wet and dry) and comparing the measured growth rates such as time to flowering, plant size, yield and similar parameters.

[00399] Tolerance to heat stress - Tolerance to heat stress is achieved by exposing plants to temperatures above 34 ° C for a certain period. The tolerance of the plant is examined after transferring the plants back to 22 ° C for recovery and evaluation after 5 days in relation to internal controls (non-transgenic plants) or plants not exposed to either cold stress or stress. heat.

[00400] Efficiency in the use of water - can be determined as the biomass produced by unit transpiration. To analyze WUE, the relative water content of the leaf can be measured in control and transgenic plants. The fresh weight (FW 1 fresh weight) is recorded immediately; then, the leaves are placed for 8 hours in distilled water at room temperature in the dark and the turgid weight (TW 1 turgid weight) is recorded. The total dry weight (DW 1 dry weight) is recorded after drying the leaves at 60 ° C at a constant weight. The relative water content (RWC 1 relative water content) is calculated according to the following Formula I:
Formula I
RWC = [(FW - DW) / (TW - DW)] x 100

[00401] Efficiency in the use of fertilizers - To analyze whether transgenic plants are more responsive to fertilizers, the plants are grown on agar plates or pots with a limited amount of fertilizer, as described, eg, in Examples 18- 20 below and in Yanagisawa et al (Proc Natl Acad Sci USA. 2004; 101: 7833-8). The plants are analyzed for their general size, time for flowering, production, protein content of the sprout and / or grain. The parameters verified are the general size of the mature plant, its wet and dry weight, the weight of the seeds generated, the average seed size and the number of seeds produced per plant. Other parameters that can be tested are: the chlorophyll content of the leaves (such as the nitrogen status of the plant and the degree of greenness of the leaf is highly correlated), the amino acid and total protein content of the seeds or other parts of the plant such as leaves or buds, oil content, etc. Similarly, instead of supplying nitrogen in limiting quantities, phosphate or potassium can be added in increasing concentrations. Again the same measured parameters are the same as those listed above. In this way, the efficiency in the use of nitrogen (NUE), the efficiency of the use of phosphate (PUE I phosphate use efficiency) and the efficiency of the use of potassium (KUE I potassium use efficiency) are evaluated, verifying the capacity of transgenic plants of develop under conditions of nutrient restriction.

[00402] Efficiency in the use of nitrogen - To analyze whether transgenic plants (eg, Arabidopsis plants) are more responsive to nitrogen, the plants are grown at 0.75-3 mM (nitrogen deficiency conditions) or 6 -10 mM (adequate nitrogen concentration). Plants are allowed to grow for an additional 25 days or until seed production. Then, the plants are analyzed for their general size, time for flowering, production, sprout and / or grain / seed protein content. The verified parameters can be the general size of the mature plant, its wet and dry weight, the weight of the seeds generated, the average size of the seed and the number of seeds produced per plant. Other parameters that can be tested are: the chlorophyll content of the leaves (such as the nitrogen status of the plant and the degree of greenness of the leaf is highly correlated), the amino acid and total protein content of the seeds or other parts of the plant like the leaves or the buds the oil content. Transformed plants that do not exhibit substantial physiological and / or morphological effects, or that exhibit higher levels of the measured parameters than wild-type plants, are identified as nitrogen-efficient plants.

[00403] Efficiency test in the use of nitrogen using seedlings - The test is carried out according to Yanagisawa-S. et al. with minor modifications ("Metabolic engineering with Dofl transcription factor in plants: Improved nitrogen assimilation and growth under low-nitrogen conditions" Proc. Natl. Acad. Sci. USA 101, 7833-7838). Briefly, transgenic plants that are grown for 7-10 days in 0.5 x MS [MurashigeSkoog] supplemented with a selection agent are transferred to two nitrogen-limiting conditions: The MS medium in which the combined nitrogen concentration (NH4NO3 and KNO3) was 0.75 mM (nitrogen deficient conditions) or 6-15 mM (adequate nitrogen concentration). The plants were allowed to grow for an additional 30-40 days and were then photographed, removed individually from the Agar (the sprout without the roots) and weighed immediately (fresh weight) for further statistical analysis. Structures for which only T1 seeds are available are sown in a selective medium and at least 20 seedlings (each representing an independent transformation event) are carefully transferred to the nitrogen-limiting medium. For structures in which T2 seeds are available, different transformation events are analyzed. Generally, 20 plants selected at random from each event are transferred to the nitrogen-limiting medium and allowed to grow for an additional 3-4 weeks and are weighed individually at the end of that period. Transgenic plants are compared to control plants grown in parallel under the same conditions. False transgenic plants expressing the uidA reporter gene (GUS) under the same promoter or transgenic plants carrying the same promoter but without a reporter gene are used as controls.

[00404] Determination of nitrogen - The procedure for determining the concentration of N (nitrogen) in the structural parts of plants involves the method of digestion of potassium persulfate to convert organic N to NO3- (Purcell and King 1996 Argon. J. 88: 111-113), the mediated reduction of Cd-modified NO3- to NO2- (Vodovotz 1996 Biotechniques 20: 390-394) and the measurement of nitrite by the Griess assay (Vodovotz 1996, supra). The absorbance values are measured at 550 nm in relation to a standard NaNO2 curve. The procedure is described in detail in Samonte et al. 2006 Agron. J. 98: 168-176.

[00405] Germination tests - Germination tests compare the percentage of seeds from transgenic plants that could complete the germination process to the percentage of seeds from the control planes that are treated in the same way. Normal conditions are considered, eg, incubations at 22 ° C under daily cycles of 22 hours of light and 2 hours of darkness. The evaluation of germination and seedling vigor is carried out between 4 and 14 days after planting. The basal medium is the 50% MS medium (Murashige and Skoog, 1962 Plant Physiology 15, 473-497).

[00406] Germination is also verified in unfavorable conditions such as cold (incubation at temperatures below 10 ° C instead of 22 ° C) or using solutions for inhibiting the seed that contain high concentrations of an osmolyte such as sorbitol (in concentrations 50 mM, 100 mM, 200 mM, 300 mM, 500 mM and up to 1000 mM) or by applying increasing concentrations of salt (from 50 mM, 100 mM, 200 mM, 300 mM, 500 mM NaCl).

[00407] The effect of the transgene on the vigor, growth rate, biomass, production and / or oil content of the plant can be determined using known methods.

[00408] Vigor of the plant - The vigor of the plant can be calculated by increasing the growth parameters such as leaf area, fiber length, rosette diameter, fresh weight of the plant and similar parameters per period.

[00409] Growth rate -The growth rate can be measured using the digital analysis of cultivated plants. For example, images of greenhouse-grown plants based on the terrain can be captured every 3 days and the area of the rosette can be calculated by digital analysis. The growth of the rosette area is calculated using the difference in the rosette area between the sampling days divided by the difference in days between the samples.

[00410] The growth rate assessment can be performed by measuring the biomass produced from the plant, the size of the leaf or the length of the root per period (can be measured in cm2 per day of leaf area).

[00411] The area of relative growth can be calculated using Formula II.
Formula II:
Area of relative growth rate = Area regression coefficient over the time cycle.

[00412] Thus, the rate of the relative growth area is in units of 1 / day and the rate of growth in length is in units of 1 / day.

[00413] Seed production -The evaluation of seed production per plant can be carried out by measuring the amount (weight or size) or quantity (that is, the number) of dry seeds produced and harvested from 8-16 plants and divided by the number of plants.

[00414] For example, the total seeds of 8-16 plants can be collected and weighed using, for example, an analytical balance, and the total weight can be divided by the number of plants. Seed production by cultivation area can be calculated in the same way taking into account the cultivation area determined for a single plant. Increasing seed production per cultivation area can be achieved by increasing seed production per plant, and / or increasing the number of plants capable of being cultivated in a given area.

[00415] In addition, seed production can be determined through the weight of 1000 seeds. The weight of 1000 seeds can be determined as follows: the seeds are spread on a glass tray and a photograph is taken. Each sample is weighed and then, using digital analysis, the number of seeds in each sample is calculated.

[00416] The 1000 weighed seeds can be calculated using Formula III:
Formula III:
Weight of 1000 Seeds = number of seeds in the sample / weight of the sample X 1000

[00417] The Harvest Index can be calculated using Formula IV.
Formula IV:
Harvest index = Average seed production per plant / Average dry weight.

[00418] Protein concentration in the grain - The protein content in the grain (g of protein in the grain m-2) is estimated as the product of the mass of grain N (g of N of m-2) multiplied by the conversion rate of N / k-5.13 protein (Mosse 1990, supra). The protein concentration in the grain is estimated as the ratio of the protein content in the grain per unit mass of the grain (g of protein in the grain kg-1 of the grain).

[00419] Length of fibers - The length of fibers can be measured using a fibrograph. The fibrograph system was used to compute the length in terms of "Upper Half Average" length. The average of the upper half (UHM 1 upper half mean) is the average length of the longest half of the fiber distribution. The fibrograph measures the length in wingspan lengths at a certain percentage point (http: // www (dot) cottoninc (dot) with / ClassificationofCotton /? Pg = 4 # Length).

[00420] According to some applications of the present patent application, the increase in corn production can be manifested as one or more of the following items: increase in the number of plants per cultivation area, increase in the number of ears per plant , increase in the number of rows per ear, number of grains per row of ear, the weight of the grain, the weight of a thousand grains (weight per 1000), length / diameter of the ear, increased oil content per grain and increased content starch per grain

[00421] As mentioned, the increase in plant production can be determined by several parameters. For example, the increase in rice production can be manifested by an increase in one or more of the following items: number of plants per cultivation area, number of panicles per plant, number of spikelets per panicle, number of flowers per panicle, increase the seed filling rate, increased weight per thousand grains (weight per 1000), increased oil content per seed, increased starch content per seed, among others. An increase in production can also result in the modified architecture, or it can occur because of the modified architecture.

[00422] Similarly, the increase in soybean production can be manifested by an increase in one or more of the following items: number of plants per cultivation area, number of pods per plant, number of seeds per pod, increased filling rate of the seed, increase in the weight of 1000 seeds (weight per 1000), reduction of the tearing of the pods, increase of the oil content per seed, increase of the protein content for the seed, among others. An increase in production can also result in the modified architecture, or it can occur because of the modified architecture.

[00423] The increase in canola production can be manifested by an increase in one or more of the following items: number of plants per cultivation area, number of pods per plant, number of seeds per pod, increase in the seed filling rate , increase in the weight of 1000 seeds (weight per 1000), reduction in the tearing of the pods, increase in the oil content per seed, among others. An increase in production can also result in the modified architecture, or it can occur because of the modified architecture.

[00424] The increase in cotton production can be manifested by an increase of one or more of the following items: number of plants per cultivation area, number of capsules per plant, number of seeds per capsule, increase in the seed filling rate , an increase in the weight of a thousand seeds (weight per 1000), an increase in the oil content per seed, an improvement in the length of the fibers, fiber resistance, among others. An increase in production can also result in the modified architecture, or it can occur because of the modified architecture.

[00425] Oil content - The oil content of a plant can be determined by extracting the oil from the seed or the plant portion of the plant. Soon, lipids (oil) can be removed from the plant (eg, seed) by grinding the plant tissue in the presence of specific solvents (eg, hexane or petroleum ether) and extracting the oil in a continuous extractor . The indirect analysis of the oil content can be performed using several methods known as Nuclear Magnetic Resonance Spectroscopy (NMR 1 nuclear magnetic resonance), which measures the resonance energy absorbed by the hydrogen atoms in the liquid state of the sample [See, p. e.g., Conway TF. and Earíe FR., 1963, Journal of the American Oil Chemists' Society; Springer Berlin / Heidelberg, ISSN: 0003-021X (Print) 1558-9331 (Online)]; the Near Infrared Spectroscopy (NI near infrared), which uses the absorption of near infrared energy (1100-2500 nm) by the sample; and a method described in W0 / 2001/023884, which is based on the extraction of oil with solvent, evaporating the solvent in a gaseous stream that forms oil particles and directing a light into the gas stream and in the oil particles, which forms detectable reflected light.

[00426] Thus, the present application for a patent for invention is of high agricultural value to promote the production of commercially desired crops (eg, biomass from a plant organ such as poplar wood, or from a reproductive organ such as number of seeds or seed biomass).

[00427] Any of the transgenic plants described above or parts of them can be processed to produce a food, feed, protein or oil preparation, as for ruminant animals.

[00428] The transgenic plants described above, which exhibit an increase in the oil content, can be used to produce vegetable oil (extracting the oil from the plant).

[00429] Vegetable oil (including seed oil and / or vegetable portion oil) produced according to the method of the present patent application can be combined with a variety of other ingredients. The specific ingredients included in a product are determined according to the intended use. Exemplary products include animal feed, raw material for chemical modification, biodegradable plastic, mixed food product, edible oil, biofuel, cooking oil, lubricant, biodiesel, snacks, cosmetics and raw materials for the fermentation process. Exemplary products to be incorporated into vegetable oil include animal feed, human food products such as extruded snacks, breads, as a food binding agent, aquaculture feed, fermentable mixtures, food supplements, sports drinks, nutritional food bars, multivitamin supplements, beverages dietary products and cereals.

[00430] According to some applications of the present patent application, the oil comprises a seed oil.

[00431] According to some applications of the present patent application, the oil comprises an oil from the plant portion (the oil from the plant portion of the plant).

[00432] According to some applications of the present patent application, the plant cell forms a part of the plant.

[00433] According to another application of the present application for a patent, a food or feed is provided, comprising the plants or a respective part of the present application for a patent.

[00434] As used herein, the term "approximately" refers to ± 10%.

[00435] The terms "comprises", "comprising", "includes", "including", "presenting" and their conjugations mean "including, but not limited to".

[00436] The term "consists of" means "including and limited to".

[00437] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, steps and / or parts do not materially alter the basic and new characteristics of the composition, method or structure claimed.

[00438] As used here, the singular form "um", "uma" and "o / a" include references in the plural, unless the context clearly specifies otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures of them.

[00439] Throughout the present application, various applications of the present invention patent application can be presented in varied format. It should be understood that the description in varying format is purely for convenience and brevity and should not be considered as an inflexible limitation on the scope of the present application for an invention patent. Therefore, the description of a variety should be considered as having specifically revealed all possible subvariations, as well as the individual numerical values within that variation. For example, the description of a variation such as 1 to 6 should be considered to have specifically revealed subvariations such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, eg, 1, 2, 3, 4, 5 and 6. This applies regardless of the range of the range.

[00440] Whenever a numerical variation is indicated in this document, this means including any quoted numeral (fractional or integral) within the indicated variation. The phrases "varying / varies between" a first indicated number and a second indicated number and "varying / varies from" a first indicated number "to" to a second indicated number are used interchangeably here and are meant to include the first and second indicated numbers and all fractional and integral numerals between them.

[00441] As used herein, the term "method" refers to ways, means, techniques and procedures for performing a given task including, but not limited to, those ways, means, techniques and procedures that are known, or readily developed from in ways, means, techniques and procedures known to professionals in the chemical, pharmacological, biological, biochemical and medical techniques.

[00442] It should be noted that certain features of the present application for an invention patent, which are, for the sake of clarity, described in the context of separate applications, can also be presented in combination in a single application. Conversely, various features of the present invention application, which are, for the sake of brevity, described in the context of a single application, can also be presented separately or in any suitable subcombination or in the appropriate manner in any other application described in the present application. of patent of invention. Certain characteristics described in the context of various applications should not be considered essential characteristics of those applications, unless the application is inoperative without these elements.

[00443] Various applications and aspects of the present patent application are outlined above and, as claimed in the claims section below, find experimental support in the following examples.

EXAMPLES

[00444] Now reference is made to the following examples which, together with the above descriptions, illustrate some applications of the present invention patent application in a non-limiting way.

[00445] Generally, the nomenclature used in this document and the laboratory procedures used in the present application for a patent include molecular, biochemical, microbiological and recombinant DNA techniques. These techniques are explained in detail in the literature. See, eg, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes 1-111 Ausubel, RM, ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies, as defined in US Patents No. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, JE, ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan JE, ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", WH Freeman and Co., New York (1980); available immunoassays are extensively described in the scientific and patent literature, see, eg, U.S. Patent No. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521;
"Oligonucleotide Synthesis" Gait, MJ, ed. (1984); "Nucleic Acid Hybridization" Hames, BD, and Higgins SJ, eds. (1985); "Transcription and Translation" Hames, BD, and Higgins SJ, Eds. (1984); "Animal Cell Culture" Freshney, RI, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set out in this document. Other general references are presented throughout this document. The procedures described in these works are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

EXPERIMENTAL AND BIOINFORMATIC METHODS IN GENERAL

[00446] RNA extraction - Fabrics grown under various growing conditions (as described below) were sampled and RNA was extracted using Invitrogen's TRIzole Reagent [http: // www (dot) invitrogen (dot) com / content (dot) cfm? pageid = 469]. Approximately 30-50 mg of tissue was collected from those sampled. The heavy tissues were crushed using liquid nitrogen pestle and mortar and resuspended in 500g1 of TRIzol Reagent. To the homogenized lysate, 100 g of chloroform were added followed by precipitation using isopropanol and two washes with 75% ethanol. The RNA was eluted in 30g1 of RNase-free water. The RNA samples were cleaned using the cleaning protocol as Qiagen's RNeasy minikit according to the manufacturer's protocol (QIAGEN Inc, CA USA). For convenience, each type of tissue with microarray expression information received an Identification of the expression set.

[00447] Correlation analysis - was performed for selected genes, according to some applications of the present patent application, in which the characterized parameters (parameters measured according to the correlation identities) were used as "X-axis" for the correlation with the tissue transcriptome that was used as the "Y axis". For each gene and parameter measured, a correlation coefficient "R" was calculated, using Ervilharson's correlation together with a p-value for the significance of the correlation. When the correlation coefficient (R) between the levels of a gene expression in a given tissue and a phenotypic performance through ecotypes / variety / hybrid is high in absolute value (between 0.5-1), there is an association between the gene (specifically the level of expression of that gene) and the phenotypic characteristic (eg, increase in production, growth rate, efficiency in the use of nitrogen, tolerance to abiotic stress and the like).

EXAMPLE 1 IDENTIFICATION OF GENES THAT INCREASE EFFICIENCY IN THE USE OF NITROGEN (NUE), EFFICIENCY IN THE USE OF FERTILIZER (FUE), PRODUCTION, GROWTH RATE, VIGOR, BIOMASS, OIL CONTENT, TOLERANCE TO ABIOTIC STRESS (ABSTANCE) AND / OR WATER USE (WUE) ON PLANTS

[00448] The present inventors have identified polynucleotides whose upward regulation of respective expression in plants increases efficiency in the use of nitrogen (NUE), efficiency in the use of fertilizers (FUE), production (eg, seed production, production oil content, quality and / or quantity of grains), growth rate, vigor, biomass, oil content, fiber production, fiber quality, fiber length, tolerance to abiotic stress ( ABST) and / or water use efficiency (WUE) of a plant.

[00449] All nucleotide sequence data sets used here were sourced from publicly available databases or from sequences obtained using Solexa technology (eg, barley and sorghum). Sequence data for 100 different plant species was entered into a comprehensive, unique database. Other information on gene expression, protein annotation, enzymes and pathways has also been incorporated. The main databases used include: Genomes

[00450] Arabidopsis genome [TAIR genome, version 6 (http: // www (dot) Arabidopsis (dot) org /)];
Rice genome [structure IRGSP 4.0 (http: // rgp (dot) dna (dot) affrc (dot) go (dot) jp / IRGSP /)];
Poplar [Populus trichocarpa, release 1.1 of JGI (joint release v1.0) (http: // www (dot) genome (dot) jgi-psf (dot) org /)];
Brachypodium [4x JGI set, http: // www (dot) brachpodium (dot) org)];
Soy [DOE-JGI SCP, version Glyma0 (http: // www (dot) phytozome (dot) net /)];
Grape [Franco-Italian Public Consortium for Characterization of the Grape Genome (http: // www (dot) genoscope (dot) ens (dot) fir /)];
Castor bean [TIGR / J, Craig Venter Institute, 4x set [(http: // msc (dot) jevi (dot) org / rcommunis];
Sorghum [DOE-JGI SCP, Sbil version [http: // www (dot) phytozome (dot) net /)];
Corn [http: // milhosequence (dot) org /];
Cucumber [http: // cucumber (dot) genomics (dot) org (dot) cn / page / cucumber / index (dot) jsp]
Tomato [http: // solgenomics (dot) net / tomato /]
Cassava [http: // www (dot) phytozome (dot) net / cassava (dot) php]

[00451] Expressed EST and mRNA sequences were extracted from the following databases:
GenBank (http: // www (dot) ncbi (dot) nlm (dot) nih (dot) gov / Genbank /);
RefSeq (Http: // www (dot) ncbi (dot) nlm (dot) nih (dot) gov / RefSeq /);
TAIR (Http: // www (dot) Arabidopsis (dot) org /); Protein databases and pathways
Uniprot [http: // www (dot) uniprot (dot) org /].

[00452] AraCyc [http: // www (dot) Arabidopsis (dot) org / biocyc / index (dot) jsp].

[00453] ENZYME [http: // expasy (dot) org / enzyme /].

[00454] The microarray data sets were downloaded from:
GEO (http://www.ncbi.nlm.nih.gov/geo/)
TAIR (http://www.Arabidopsis.org/).

[00455] Data from microarrays of exclusive property (VideWO2008 / 122980 and Examples 3-10 below).

QTL and SNPs information

[00456] Gramene [http: // www (dot) gramene (dot) org / qt1 /]. Panzea [http: // www (dot) panzea (dot) org / index (dot) html].

[00457] Soy QTL: [http: // www (dot) soybeanbreederstoolbox (dot) com /].

[00458] Database Set - was designed to constitute a broad, rich, detailed, reliable and easy to analyze database, comprising genomic sequences of mRNA, EST's, DNA, publicly available, data from various cultures, as well as data gene expression, annotation and protein path, QTL's data and other relevant information.

[00459] The set of databases comprises a toolbox for improvement, structuring, genetic annotation and analysis tools that allow the construction of a tailor-made database for each genetic discovery project. The genetic enhancement and structuring tools (a) allow to reliably detect assembled variants and antisense transcripts, generating the understanding of several potential phenotypic results of a single gene. The capabilities of Compugen LTD's "LEADS" platform to analyze the human genome have been confirmed and accepted by the scientific community [see, eg, "Widespread Antisense Transcription", Yelin, et al. (2003) Nature Biotechnology 21, 379-85; "Splicing of Alu Sequences", Lev-Maor, et al. (2003) Science 300 (5623), 1288-91; "Computational analysis of alternative splicing using EST tissue information", Xie H et al. Genomics 2002] and proved to be more efficient in plant genomics, too.

[00460] Genetic set and EST grouping - For the genetic grouping and the grouping of organisms with available data of the genomic sequence (Arabidopsis, rice, castor, grape, brachypodium, poplar, soy, sorghum), the genomic version (GANG) was used LEADS. This tool allows more precise grouping of ESTs and mRNA sequences in the genome and predicts the genetic structure, as well as alternative events of grouping and antisense transcription.

[00461] For organisms without complete data of available genomic sequence, the clustering software "expressed LEADS" was applied.

[00462] Genetic annotation - Predicted genes and proteins were noted as follows: The search for sequence comparison [http: // blast (dot) ncbi (dot) nlm (dot) nih (dot) gov / Blast (dot) cgi] against all UniProt of the plant [http: // www (dot) uniprot (dot) org /] was carried out. Open reading structures of each putative transcript were analyzed and the longest ORF with the largest number of homologues was selected as the predicted protein of the transcript. The predicted proteins were analyzed by InterPro [http: // www (dot) ebi (dot) ac (dot) uk / interpro /].

[00463] The comparison against proteins from the AraCyc and ENZYME databases was used to map the predicted transcripts with the AraCyc pathways.

[00464] The predicted proteins of different species were compared using the comparison algorithm [http: // www (dot) ncbi (dot) nlm (dot) nih (dot) gov / Blast (dot) cgi] to validate the accuracy of predicted protein sequence and for the efficient detection of orthologists.

[00465] Gene expression profile - Several data sources have been explored regarding the gene expression profile, namely microarray data and digital expression profile (see below). According to the gene expression profile, a correlation analysis was performed to identify genes that are co-regulated under different stages of development and environmental conditions and associated with different phenotypes.

[00466] Publicly available microarray data sets were downloaded from the TAIR and NCBI GEO sites, renormalized and integrated into the database. The expression profile is one of the most important resource data for identifying important genes for production.

[00467] A digital summary of the expression profile was compiled for each grouping according to all keywords included in the sequence records comprising the grouping. Digital expression, also known as electronic Northern Blot, is a tool that displays the virtual profile of the expression based on the EST sequences that form the genetic grouping. The tool presents the expression profile of a cluster in terms of the anatomy of the plant (eg, the tissue / organ in which the gene is expressed), the stage of development (the stages of development in which a gene can be found ) and the treatment profile (shows the physiological conditions under which a gene is expressed, such as drought, cold, pathogen infection, etc.). Given the random distribution of ESTs in the different clusters, the digital expression has a probability value that describes the probability that a cluster will have a total of N ESTs to contain X ESTs from a given library collection. For the probability calculations, the following is taken into account: a) the number of ESTs in the cluster, b) the number of ESTs from the involved and related libraries, c) the general number of available ESTs representing the species. In this way, clusters with low probability values are highly enriched with ESTs from the group of libraries of interest indicating a specialized expression.

[00468] Recently, the accuracy of this system was demonstrated by Portnoy et al., 2009 (Analysis Of The Melon Fruit Transcriptome Based On 454 Pyrosaquencing) in: XVII Conference on Animal and Vegetable Genomes, San Diego, CA. The transcriptomic analysis based on the relative abundance of EST's in the data was performed by pyrosequencing 454 of cDNA representing the melon mRNA. Fourteen double-stranded cDNA samples obtained from two genotypes, two fruit tissues (pulp and peel) and four developmental stages were sequenced. The GS FLX pirosaquencing (Roche / 454 Life Sciences) of non-normalized and purified cDNA samples yielded 1,150,657 express sequence tags (ESTs) that were grouped into 67,477 unigenes (32,357 singletons and 35,120 contigs). The analysis of the data obtained against the Cucurbitaceae Genomic Database [http: // www (dot) icugi (dot) org /] confirmed the accuracy of the sequencing and clustering. Expression patterns of selected genes fit their qRT-PCR (reverse transcription polymerase chain reaction) data well.

[00469] Overall, 215 genes have been identified as having a major impact on efficiency in the use of nitrogen, efficiency in the use of fertilizers, production (eg, seed production, oil production, quality and / or quantity of grains ), growth rate, vigor, biomass, oil content, fiber production, fiber quality, fiber length, tolerance to abiotic stress and / or water use efficiency when their respective expression is increased in plants. The identical genes, their cured polynucleotides and polypeptide sequences, as well as their updated sequences, according to the GenBank database, are summarized in Table 1, below.

Table 1

Tabela 1: "Polip." = polipeptídeo; "Polin." = polinucleotideo.[00470] Polynucleotides identified to increase efficiency in the use of nitrogen, efficiency in the use of fertilizers, production, growth rate, vigor, biomass, oil content, fiber production, fiber quality, fiber length, abiotic stress tolerance and / or efficiency in using a plant's water.

Table 1: "Polip." = polypeptide; "Polin." = polynucleotide.

EXAMPLE 2 IDENTIFICATION OF HOMOLOGICAL SEQUENCES THAT INCREASE EFFICIENCY IN THE USE OF NITROGEN, EFFICIENCY IN THE USE OF FERTILIZERS, PRODUCTION, GROWTH RATE, VIGOR, BIOMASS, OIL CONTENT, TOLERANCE IN ABIOTIC STRESS AND / OR EFFICIENCY.

[00471] The concepts of orthology and paralogy have recently been applied to characterizations and functional classifications on the scale of comparisons of the complete genome. Orthologists and parallels are two main types of counterparts: The former evolved from a common ancestor by specialization and the latter are related by duplication events. It is assumed that parallels arising from events of ancient duplication tend to have diverged in function while true orthologists are more likely to retain the identical function over evolutionary time.

[00472] For further investigation and identification of putative orthologists of genes that affect nitrogen use efficiency, fertilizer use efficiency, production (eg, seed production, oil production, quality and / or quantity of grains ), growth rate, vigor, biomass, oil content, tolerance to abiotic stress and / or water use efficiency, all sequences were aligned using the Basic Local Alignment Search Tool / ). Sufficiently similar strings had been tentatively grouped together. These putative orthologists were further organized under a Filogram - a branching diagram (tree) assumed as a representation of the evolutionary relationships between biological taxa. Putative orthological groups were analyzed for their agreement with the filogram and, in cases of divergence, these orthological groups were divided appropriately.

[00473] Expression data was analyzed and EST libraries were classified using a fixed vocabulary of personalized terms, such as stages of development (for example, genes that show the similar expression profile, through upward-regulated development at the specific stage, as in the grain storage phase) and / or plant organ (for example, genes that show a similar expression profile in their organs with upward regulation in specific organs, such as the seed). The annotations of all ESTs grouped to a gene were analyzed statistically by comparing their frequency in the set in relation to their abundance in the database, allowing the structure of a numerical and graphical expression profile of that gene, which is called " digital expression ". The logic of using these two complementary methods with methods of studying phenotypic association of QTL's, SNPs and phenotypic expression is based on the assumption that true orthologists tend to retain the identical function over evolutionary time. These methods provide different sets of indications about the functional similarities between two homologous genes, similarities at the level of the amino acid sequence - identical in the protein domains and similarity in expression profiles.

[00474] Searching and identifying homologous genes involves tracking available sequence information, for example, in public databases that include, but are not limited to, the Japan DNA Database (DDBJ 1 DNA Database of Japan) , Genbank and the Nucleic Acid Sequence Database of the Nuclear Biology Laboratory (EMBL 1 European Molecular Biology Laboratory) or their versions or the MIPS database. A number of different search algorithms have been developed, including, but not limited to, the set of programs referred to as BLAST programs. There are five implementations of BLAST, three designed for nucleotide sequence queries (BLASTN, BLASTX and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology: 76-80, 1994; Birren et al., Genome Analysis, I: 543, 1997). Such methods involve aligning and comparing the sequences. The BLAST algorithm calculates the percentage of sequence identity and performs a statistical analysis of similarity between the two sequences. The software for performing BLAST analysis is available to the public through the National Biotechnology Information Center. Others of these types of software or algorithms are GAP, BESTFIT, FASTA and TFASTA. GAP uses the Needleman and Wunsch algorithm (J. Mol. Biol. 48: 443-453, 1970) to find the alignment of two complete strings that maximizes the number of matches and minimizes the number of gaps.

[00475] Homologous genes may belong to the same genetic family. The analysis of a genetic family can be performed using sequence similarity analysis. To perform this analysis, standard programs for multiple alignments can be used, for example, Clustal W. A Neighbor Joining tree of the neighboring proteins of the genes homologous to the genes of some applications of the present patent application can be used to provide an overview of structural and ancestral relationships. The sequence identity can be calculated using an alignment program as described below. Other plants are expected to have a similar functional gene (ortholog) or a family of similar genes and such genes will provide the same preferred phenotype as the genes presented here. Advantageously, these members of the family may be useful in the methods of some applications of the present application. Examples of other plants include, but are not limited to, barley (Hordeum vulgare), Arabidopsis (Arabidopsis thaliana), Maize (Zea mays), Cotton (Gossypium), Canola (Brassica napus), Rice (Oryza sativa), Cane sugar (Saccharum officinarum), Sorghum (Sorghum bicolor), Soy (Glycine max), Sunflower (Helianthus annuus), Tomato (Lycopersicon esculentum) and Wheat (Triticum aestivum).

[00476] The aforementioned analyzes for a sequence homology are preferably performed on a full length sequence, but can also be based on a comparison of certain regions, such as conserved domains. The identification of such domains would also be good within the knowledge of a person skilled in the art and would involve, for example, a computer-readable format of the nucleic acids for some applications of the present patent application, the use of software programs for alignment and use of publicly available information on protein domains, conserved motifs and boxes This information is available in the PRODOM database (http: // www (dot) biochem (dot) ucl (dot) ac (dot) uk / bsm / dbbrowser / protocol / prodomqry (dot) html), PIR (http: // pir (dot) Georgetown (dot) edu /) or Pfam (http: // www (dot) sanger (dot) ac (dot) uk / Software / Pfam /). Sequence analysis programs developed for motif research can be used to identify conserved fragments, regions and domains as mentioned above. Preferred computer programs include, but are not limited to, MEME, SIGNALSCAN and GENESCAN.

[00477] A person skilled in the art can use the homologous sequences provided here to find similar sequences in other species and other organisms. The homologues of a protein include peptides, oligopeptides, polypeptides, proteins and enzymes that have amino acid substitutions, deletions and / or insertions in relation to the unmodified protein in question and that have biological and functional activity similar to the unmodified protein from which are derived. To produce such homologues, the amino acids in the protein can be replaced by other amino acids that have similar properties (conservative modifications, such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form a structure or break helical structures or 3-leaf structures). Conservative substitution tables are well known in the art (see, eg, Creighton (1984) Proteins. W.H. Freeman and Company). Homologues of an amino acid encompass nucleic acids with nucleotide substitution, deletions and / or insertions in relation to the unmodified nucleic acid in question and which have biological and functional activity similar to the unmodified nucleic acid from which they are derived.

[00478] Polynucleotides and polypeptides with significant homology to the identified genes described in Table 1 (Example 1, above) were identified from the databases, using the BLAST software with the Blastp and tBlastn algorithms as filters for the first phase and the needle (EMBOSS package) or FRAME alignment + algorithm for the second phase. The local identity (Blast alignments) was defined with a very permissive cut - 60% of Identity in a range of 60% of the sequence lengths because it uses only one filter for the global alignment phase. The default filtering of the Blast package was not used (setting the parameter "-F F").

[00479] In the second stage, the homologues were defined based on the global identity of at least 80% for the main genetic polypeptide sequence.

[00480] Two distinct ways to find the ideal global alignment for protein or nucleotide sequences were used in this application.

[00481] Between two proteins (after the blastp filter):
Needleman-Wunsch EMBOSS- 6.0.1 algorithm with the following modified parameters: gapopen = 8 gapextend = 2. The rest of the parameters remained unchanged from the standard options described above.

[00482] Between a protein sequence and a nucleotide sequence (after the rblastn filter):
GenCore 6.0 OneModel application, using the Frame + algorithm with the following parameters: model = frametp2n.model mode = gglobal - q = protein.sequence -db = nucleotide.sequence. The rest of the parameters remained unchanged from the standard options described above.

[00483] The query polypeptide sequences were SEQ ID NO: 470-762 (which are encoded by SEQ ID NO: 1- 469 polynucleotides, shown in Table 1, above) and the orthologous and homologous sequences identified having at least 80% of the global sequence identity is provided in Table 2, below. These homologous genes (eg, orthologists) are expected to increase NUE, production, seed production, oil production, oil content, growth rate, fiber production, fiber quality, biomass, vigor and / or ABST of a plant.

Table 2

[00484] Homologues of the identified genes / polypeptides to increase efficiency in the use of nitrogen, efficiency in the use of fertilizer, production, seed production, growth rate, vigor, biomass, oil content, fiber production, fiber quality, fiber length, tolerance to abiotic stress and / or water use efficiency of a plant.

[00485] Table 2: Homologous polypeptides and polynucleotides of the genes identified in Table 1 and their cloned genes are provided, which can increase the efficiency in the use of nitrogen, efficiency in the use of fertilizer, production, seed production, growth rate, vigor, biomass, oil content, fiber production, fiber quality, fiber length, tolerance to abiotic stress and / or efficiency in the use of water from a plant. Homology was calculated as% identity over the alignment sequences. The query strings were the polypeptide sequences of SEQ ID NO: 70-762 and the sequences in question are polypeptide sequences or polynucleotide sequences that were dynamically transposed in all six reading frames identified in the database based on identity greater than 80% with respect to the polypeptide sequences of the consultation. "Polip." = polypeptide; "Polin" - polynucleotide; "Algor." = Algorithm; "globlastp" - global homology using blastp; "glotblastn" - global homology using tblastn. "Hom." - counterpart.

[00486] The result of the functional genomic approach described here is a set of genes with high estimates of improved efficiency in the use of nitrogen, efficiency in the use of fertilizer, production, seed production, growth rate, vigor, biomass, content of oil, fiber production, fiber quality, fiber length, tolerance to abiotic stress and / or efficiency in the use of water from a plant, increasing its expression.

[00487] Although each gene is estimated to have its own impact, it is expected that modifying the mode of expression of more than one gene or gene product (RNA, polypeptide) will provide an additive or synergistic effect on a desired characteristic ( eg efficiency in the use of nitrogen, efficiency in the use of fertilizer, production, growth rate, vigor, biomass, oil content, tolerance to abiotic stress and / or efficiency in the use of water in a plant). By altering the expression of each gene described here, the gene alone or the set of genes increases overall production and / or other important agricultural traits and, thus, is expected to increase agricultural productivity.

EXAMPLE 3 PRODUCTION OF ARABIDOPSIS TRANSCRIPTOMA AND HIGH PRODUCTIVITY CORRELATION ANALYSIS USING ARABIDOPSIS 44K OLIGONUCLEOTIDE MICRO-ARRANGEMENT.

[00488] To produce a high productivity correlation analysis comparing plant phenotype and gene expression level, the present inventors used an Arabidopsis oligonucleotide microarray, produced by Agilent Technologies [http: // www (dot) chem (dot) agilent (dot) com / Scripts / PDS (dot) asp? 1Page = 50879]. The matrix oligonucleotide represents about 44,000 Arabidopsis transcription genes. To define the correlations between the levels of RNA expression with NUE, the parameters related to the components of production or vigor, several characteristics of plants of 14 different Arabidopsis ecotypes were analyzed. Among them, ten ecotypes encompassing the observed variation were selected for the analysis of RNA expression. The correlation between the RNA levels and the characterized parameters was analyzed using Pearson correlation test [Http: // www (dot) davidmlane (dot) com / hyperstat / A34739 (dot) html].

Experimental Procedures

[00489] Arabidopsis tissues analyzed - Two plant tissues [leaves and stems] growing at two different levels of nitrogen fertilization (1.5 mM Nitrogen or 6 mM Nitrogen) were sampled and RNA was extracted, as described above. Each type of microarray expression information tissue received a set ID, as summarized in Table 3 below.
Table 3
Arabidopsis transcribtoma experimental sets

[00490] Evaluation of parameters related to Arabidopsis production and vigor components at different levels of nitrogen fertilization - 10 accessions of Arabidopsis in 2 repetitive lots, each containing 8 plants per lot, were grown in the greenhouse. The cultivation protocol used was as follows: seeds sterilized on the surface were sown in Eppendorf tubes containing Murashige-Skoog x 0.5 basal salt medium and grown at 23 'C in daily cycles of 12 hours of light and 12 hours in the dark for 10 days. Then, seedlings of similar size were carefully transferred to pots filled with a mixture of perlite and peat in a 1: 1 ratio. The constant nitrogen limiting conditions were achieved by irrigating the plants with a solution containing 1.5mM organic nitrogen in the form of KNO3, supplemented with 2mM CaCl2, 1.25mM KH2PO4, 1.50mM MgSO4, 5mM KC1, H3B03 at 0.01 mM, and microelements, while normal irrigation conditions were achieved by applying a 6mM inorganic nitrogen solution also in the form of KNO3, supplemented with 2mM CaC12, 1.25mM KH2PO4, 1.50mM MgSO4, 0.01 mM H3B03 and microelements. To accompany the growth of the plant, trays were photographed on the day that the limiting conditions were initiated and subsequently every 3 days for about another 15 days. The rosette plant area was then determined from digital photos. The ImageJ software was used to quantify the plant size from digital photos [Http: // rsb (dot) info (dot) nih (dot) gov / ij /] using exclusive scripts designed to analyze the size of the rosette area from individual plants as a function of time. The image analysis system includes a personal computer (Intel P4 3.0 Ghz processor) and a public domain program - ImageJ 1.37 (Java based on the image processing program, which was developed at the National Institutes of Health in the United States and freely available on the internet [Http: // rsbweb (dot) nih (dot) gov /]). Then, analysis data were saved to text files using the statistical analysis software JMP (instituto SAS).

[00491] The data parameters collected are summarized in Table 4, below.
Table 4
Parameters related to Arabidopsis (vectors)

[00492] Table 4 "N" = Nitrogen in the noted concentrations; "gr." = grams; "SPAD" = chlorophyll levels; "t50" = time when 50% of the plants flowered; "gr./ SPAD unit" = plant biomass expressed in grams per unit of nitrogen in the plant measured by SPAD. "DW" = dry weight of the plant; "N / DW level" = plant nitrogen level measured in SPAD unit by plant biomass [gr .; "DW / level N" plant biomass per plant [gr.] / SPAD unit;
Evaluation of NUE, parameters related to the components of production and vigor - Ten ecotypes of Arabidopsis were grown in trays, each containing 8 plants per lot, in a greenhouse with controlled temperature conditions for about 12 weeks. The plants were irrigated with a different nitrogen concentration as described above depending on the treatment applied. During that time, data was collected, documented and analyzed. Most of the parameters chosen were analyzed by digital image formation.

Digital Image Formation - Essay in the Greenhouse

[00493] An image acquisition system, consisting of a digital reflex camera (Canon EOS 400D) attached with a 55 mm focal length lens (Canon EF-S series) placed in a custom aluminum mount, was used to capture images of plants planted in an environmentally controlled greenhouse. The image capture process was repeated every 2 and 3 days starting on the 9th to 12th until the 16th to the 19th (respectively) from the transplant.

[00494] An image analysis system was used, which consists of a personal computer (Intel P4 3.0 Ghz processor) and a public domain program - ImageJ 1.37, Java based on the image processing program, which was developed in the National Institutes of Health of the United States and freely available on the internet at Http: // rsbweb (dot) nih (dot) gov /. The images were captured at a resolution of 10 Mega Pixels (3888x2592 pixels) and stored in a low compression JPEG format (standard Joint Photographic Experts Group). Then, processing output data was saved to text files and analyzed using the statistical analysis software JMP (instituto SAS).

[00495] Leaf analysis - Using digital analysis, leaf data was calculated, including leaf number, leaf blade area, rosette diameter and rosette area.

[00496] Plant growth rate: the relative growth rate (RGR) of the leaf blade area (Formula V), leaf number (Formula VI), rosette area (Formula VII), rosette diameter (Formula VIII), lot coverage (Formula IX) and Petiole Relative Area (Formula X) were calculated as follows:
Formula V
Relative growth rate of leaf blade area = regression coefficient of leaf area over time.
Formula VI
Relative growth rate of the leaf number of the plant = regression coefficient of the leaf number of the plant over time.
Formula VII
Relative growth rate of the rosette area = regression coefficient of the rosette area over time.
Formula VIII Rate of relative growth of the diameter of the rosette = regression coefficient of the diameter of the rosette over time.
Formula IX
Relative growth rate of lot coverage = lot regression coefficient.
Formula X
Relative Petiole Area = [(leaf blade * leaf number) / Rosette.

[00497] Seed production and weight of 1000 seeds - At the end of the experiment, all seeds from all lots were collected and measured in order to measure seed production per plant in terms of total seed weight per plant (gr. ). To calculate the weight of 1000 seeds, an average weight of 0.02 grams was measured from each sample, the seeds were spread on a glass tray and a picture was taken. Using digital analysis, the number of seeds in each sample was calculated.

[00498] Dry weight and seed production - At the end of the experiment, the plant was harvested and left to dry at 30 ° C in a drying chamber. The biomass was separated from the seeds, weighed and divided by the number of plants. Dry weight = total weight of the plant part above the ground (excluding roots) after drying at 30 ° C in a drying chamber.

[00499] Harvest Index -The harvest index was calculated using Formula IV, as described above.

[00500] T50 days for flowering - Each repetition was monitored for the flowering date. The flowering days were calculated from the date of sowing up to 50% of the flowering lots.

[00501] Nitrogen level in the plant - The chlorophyll content of the leaves is a good indicator of the state of nitrogen in the plant since the degree of greenness of the leaf is highly correlated to this parameter. The chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and the measurement was performed at the time of flowering. The SPAD measurement readings were taken on a new, fully developed sheet. Three measurements per sheet were taken per batch. Based on this measurement, the parameters, such as proportion between seed production per unit of nitrogen [seed production / level N = seed production per plant [gr.] / Unit SPAD], DW plant per unit of nitrogen [DW / level N = plant biomass per plant [gr.] / unit SPAD], and nitrogen level per gram of biomass [level N / DW = unit SPAD / plant biomass per plant (gr.)] were calculated.

[00502] Percentage reduction in seed production - measures the amount of seeds obtained from plants when grown under nitrogen limiting conditions compared to the production of seed produced at normal nitrogen levels expressed in%.

Experimental Results

[00503] 10 different accessions of Arabidopsis (ecotypes) were cultivated and characterized by 37 parameters, as described above. The average for each of the measured parameters was calculated using the JMP software and the values are summarized in Table 5 below. The subsequent correlation analysis between the various transcriptome sets (Table 3) and the measured parameters was conducted. Then, the results were integrated into the database.
Table 5
Parameters measured in Arabidopsis accesses

[00504] Table 5 The parameters measured under various treatments in various ecotypes (Arabidopsis accessions) are provided.

EXAMPLE 4 PRODUCTION OF ARABIDOPSIS TRANSCRIPTOM AND HIGH PRODUCTIVITY CORRELATION ANALYSIS OF PARAMETERS RELATED TO PRODUCTION, BIOMASS AND / OR VIGOR USING TOTAL ARABIDOPSIS 44K OLIGONUCLEOTIDE MICRO-ARRANGEMENT

[00505] To produce a high productivity correlation analysis, comparing the plant phenotype and the level of expression of the gene, the present inventors used an oligonucleotide microarray Arabidopsis thaliana, produced by Agilent Technologies [http: // www ( dot) agilent chem (dot) com / Scripts / PDS (dot) asp? 1Page = 50879]. The array's oligonucleotide represents about 40,000 A. thaliana genes and transcripts designed based on data from the TIGR ATH1 database, v.5, and Arabidopsis MPSS databases (University of Delaware). To define the correlations between the levels of RNA expression and the parameters related to the production components, biomass or vigor, several characteristics of the plant of 15 different ecotypes Arabidopsis were analyzed. Among them, nine ecotypes encompassing the observed variation were selected for analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using the Pearson correlation test [http: // www. (Dot) davidmlane (dot) com / hyperstat / A34739 (dot) html].

Experimental Procedures

[00506] Analyzed Arabidopsis Tissues - Five tissues in different stages of development, including root, leaf, flower in anthesis, seed in 5 days after flowering (DAF 1 days after flowering) and seed in 12 DAF, representing different plant characteristics , were proven and the RNA was extracted, as described above. Each type of microarray expression information tissue received a Set ID, as summarized in Table 6 below.
Table 6
Tissues used for Arabidopsis transcription expression sets

[00507] Table 6: The identification numbers (ID) of each of the Arabidopsis expression sets (A-E) are provided. DAF [days after flowering] = days after flowering.

[00508] Evaluation of the parameters related to the components of production and vigor - Eight of the nine ecotypes of Arabidopsis were used in each of the 5 repetitive blocks (named A, B, C, D and E),
each containing 20 plants per lot. The plants were grown in a greenhouse under controlled conditions at 220C and N: P: K fertilizer (20:20:20; weight proportions) [nitrogen (N), phosphorus (P) and potassium (K)] was added. During that time, data was collected, documented and analyzed. Additional data were collected through the sowing stage of plants grown in a tissue culture on transparent agar plates of vertical cultivation. Most of the chosen parameters were analyzed by digital image formation.

[00509] Digital Image in Tissue Culture - A laboratory image acquisition system was used to capture images of shoots cut on square agar plates. The image acquisition system consists of a digital reflex camera (Canon EOS 300D) attached to a 55 mm focal length lens (Canon EF-S series), mounted on a reproduction device (Kaiser RS), which included 4 light units (4x150 Watt lamps) and located in a dark room.

[00510] Greenhouse Digital Image - The image capture process was repeated every 3 to 4 days, starting on the 7th until the 30th. The same camera attached to a 24mm focal length lens (Canon EF series) , placed in a custom iron mount, was used to capture images of larger cut plants in white bathtubs in an environmentally controlled greenhouse. The white bathtubs were square in shape, measuring 36 x 26.2 cm and 7.5 cm deep. During the capture process, the bathtubs were placed at the foot of the iron assembly, avoiding direct sunlight and casting of shadows. This process was repeated every 3 to 4 days for up to 30 days.

[00511] An image analysis system was used, consisting of a personal computer (Intel P43.0 GHz processor) and a public domain program - ImageJ 1.37, Java based on the image processing program, which was developed at the Institutes National Health Service and is freely available on the internet at http: // rsbweb (dot) nih (dot) gov /. The images were captured in a resolution of 6 Mega Pixels (3072 x 2048 pixels) and stored in a low compression JPEG format (standard of the Joint Group of Experts in Photography I Joint Photographic Experts Group). Then, the analyzed data were saved in text files and processed using the statistical analysis software JMP (instituto SAS).

[00512] Sheet analysis -Using digital analysis, the sheet data was calculated, including the sheet number, area, perimeter, length and width. On day 30, 3 to 4 representative plants were chosen from each batch of blocks A, B and C. The plants were dissected, each leaf was separated and was inserted between two glass trays, a photo of each plant was taken and the various parameters (such as total sheet area, laminar length, etc.) were calculated from the images. The circularity of the blade was calculated as the laminar width divided by the laminar length.

[00513] Root analysis -
For 17 days, the different ecotypes were grown on transparent agar plates. The plates were photographed every 3 days, starting on the 7th in the photography room and the development of the roots was documented (see examples in Figs. 3A-F). The rate of root growth was calculated according to Formula XI.
Formula XI:
Relative growth rate of root cover = Regression coefficient of root cover over time.

[00514] Analysis of the vegetative growth rate - was calculated according to Formulas V-X above. The analysis was completed with the appearance of overlapping plants.

[00515] For comparison between ecotypes, the calculated rate was normalized using the plant's development stage as represented by the number of true leaves. In cases where the 8-leaf plants were sampled twice (for example, on day 10 and day 13), only the largest sample was chosen and added to the Anova comparison.

[00516] Seed analysis in silicas - On day 70, 15 to 17 silicas were collected from each lot in blocks D and E. The silicas chosen were light brown in color, but still intact. The silikas were opened in the photography room and the seeds were spread on a glass tray, a high-resolution digital Fig was taken for each batch. Using the images, the number of seeds per siliqua was determined.

[00517] Average seed weight - At the end of the experiment, all seeds from the lots in blocks A-C were collected. An average weight of 0.02 grams was measured for each sample, the seeds were spread on a glass tray and a picture was taken. Using digital analysis, the number of seeds in each sample was calculated.

[00518] Percentage of oil in the seeds - At the end of the experiment, all seeds from the lots in blocks A-C were collected. Columbia seeds from 3 batches were mixed with the soil and then mounted in the extraction chamber. 210 ml of n-Hexane (Cat No. 080951 Biolab Ltd.) was used as the solvent. The extraction was carried out for 30 hours in an average heat of 50 ° C. Once the extraction has been completed, the nHexane was evaporated using the evaporator at 35 ❑C and vacuum conditions. The process was repeated twice. The information obtained from the Soxhlet extractor (Soxhlet, F. Die gewichtsanalytische Bestimmung des Milchfettes, Polytechnisches J. (Dingler's) 1879, 232, 461) was used to create a calibration curve for Low Resonance NMR. The oil content of all seed samples was determined using Low Resonance NMR (MARAN Ultra-Oxford Instrument) and its MultiQuant software package.

[00519] Siliqua length analysis - On day 50 since sowing, 30 silicas from different plants in each lot were sampled in block A. The silicas chosen were yellow-green and were collected from the lower parts of a stem of the cultivated plant. A digital photograph was taken to determine the length of the silica.

[00520] Dry weight and seed production - On day 80 since sowing, the plants in blocks A-C were harvested and allowed to dry at 30 ❑C in a drying chamber. The weight of the biomass and seed of each lot was separated, measured and divided by the number of plants. Dry weight = total weight of the vegetative part above the ground (excluding roots) after drying at 30 ❑C in a drying chamber; Seed production per plant = total seed weight per plant (g).

[00521] Oil production - Oil production was calculated using Formula XII.
Formula XII:
Production of seed oil = Production of seed per plant (gr.) *% Of oil in the seed.

[00522] Harvest index (seed) - The harvest index was calculated using Formula IV (described above).

Experimental Results

Tabela 7. São fornecidos os parâmetros correlacionados à Arabidopsis (ID de Correlação N° 1-18). Abreviações: Cm = centímetro(s); g = grama(s); mg = miligrama(s).[00523] Nine different Arabidopsis ecotypes were cultivated and characterized by 18 parameters (named as vectors). Table 7 describes the correlated parameters of Arabidopsis. The mean for each of the measured parameters was calculated using the JMP software (Tables 8-9) and a subsequent correlation analysis was performed. The results were then integrated into the database.
Table 7
Parameters related to Arabidopsis (vectors)

Table 7. The parameters related to Arabidopsis (Correlation ID No. 1-18) are provided. Abbreviations: Cm = centimeter (s); g = gram (s); mg = milligram (s).

[00524] The characterized values are summarized in Tables 8 and 9 below.
Table 8
Parameters measured in Arabidopsis ecotypes

[00525] Table 8. The values for each of the parameters measured in the Arabidopsis ecotypes are provided: 15 = seed production per plant (g); 16 = oil production per plant (mg); 12 =% of oil per seed; 11 = weight of 1000 seeds (g); 5 = dry matter per plant (g); 17 = harvest index; 10 = total leaf area per plant (cm); 13 = seeds per silica; 14 = length of the silica (cm).
Table 9
Additional parameters measured in Arabidoosis ecotibs

[00526] Table 9: Values are provided for each of the parameters measured in the Arabidopsis ecotypes: 6 = Plant growth rate (cm2 / day) up to 8 genuine leaves; 3 = relative root growth (cm / day) (day 13); 2 = Root length on day 7 (cm); 1 = Root length on day 13 (cm); 4 = fresh weight per plant (g) in the fixation stage; 9. = blade length (cm); 8 = Blade width (cm); 18 = Length / width of the sheet; 7 = Blade circularity.

EXAMPLE 5 DEVELOPMENT OF THE FIBER OF THE PLANT IN THE PRODUCTION OF COTTON TRANSCRIPTOMA COTTON AND HIGH PRODUCTIVITY CORRELATION ANALYSIS USING COTTON OLIGONUCLEOTIDE MICRO-ARRANGEMENT

[00527] In order to produce a high productivity gene expression correlation analysis, the present inventors used the cotton oligonucleotide microarray designed and produced by "Comparative Evolutionary Genomics of Cotton"
[http: // cottonevolution (dot) info /). This Cotton Oligonucleotide Microarray is made up of 12,006 Integrated DNA Technologies (IDT Integrated DNA Technologies) oligonucleotides derived from a set of more than 180,000 Gossypium ESTs sequenced from 30 cDNA libraries. For further details, see PCT / IL2005 / 000627 and PCT / IL2007 / 001590, which are incorporated herein in their entirety by reference.
Table 10
Cotton transcriptome experimental sets

[00528] Table 10. Cotton transcriptome expression sets are provided. "5d" = 5 days after anthesis; "10d" = 10 days after anthesis; "15d" = 15 days after anthesis. "DPA [days-post-anthesis]" = days after anthesis.

[00529] In order to define the correlations between the levels of RNA expression and the length of the fiber, fibers from 8 different cotton lines were analyzed. These fibers were selected with very good fiber quality and a high index of fila (types of Pima, originated from other cotton species, named G. barbadense), different levels of quality and index of fila from different lines of cotton. G. hirsutum: good quality and high index of thread (type Acala), and low quality and low index of thread (type Tamcot, and old varieties). A summary of the fiber length of the different lines is provided in Table 11 below.

Experimental Procedures

[00530] RNA extraction - Fiber development stages, representing different fiber characteristics, in 5, 10 and 15 DPA, were sampled and the RNA was extracted, as described above.

[00531] Fiber length assessment - The fiber length of the cotton threads was measured using fibrography. The fibrography system was used to calculate the length in terms of "Upper Half Average" length. The upper half average (UHMtupper half mean) is the average length of the longest half of the fiber distribution. Fibrography measures the length in span lengths at a given percentage point in
www (dot) cottoninc (dot) com / ClassificationofCotton /? Pg = 4 # Length].

Experimental Results

[00532] Eight different lines of cotton were grown and their fiber lengths were measured. The UHM values of the fibers are summarized in Table 11 below. The squared OR was calculated for each of the genes.
Table 11
Summary of the fiber length of the 8 different cotton threads.

[00533] Table 11: The averages and standard deviations (STD) of 8 different cotton lines are shown.

EXAMPLE 6 PRODUCTION OF RICE TRANSCRIPTOMA USING A 44K RICE OLIGONUCLEOTIDE MICRO-ARRANGEMENT

[00534] To produce a differential expression analysis of rice plants subject to nitrogen-limiting conditions compared to normal (non-limiting) nitrogen conditions, the present inventors used a Rice oligonucleotide microarray, produced by Agilent Technologies [http: / / www (dot) chem.
(dot) agilent (dot) com / Scripts / PDS (dot) asp? 1Page = 50879]. The matrix oligonucleotide represents about 44,000 rice genes and transcripts.

Experimental Procedures

[00535] Evaluation of rice plants grown at different levels of nitrogen fertilization - Five accessions of rice were grown in 3 repetitive lots, each containing 10 plants, in a greenhouse with net under semi-hydroponic conditions. In a nutshell, the cultivation protocol was as follows: The rice seeds were sown in trays filled with a mixture of vermiculite and peat in a 1: 1 ratio. The constant nitrogen limiting conditions were achieved by irrigating the plants with a solution containing 0.8mM inorganic nitrogen in the form of KNO3, supplemented with 1mM KH2PO4, 1mM MgSO4, 3.6mM K2SO4 and microelements, while normal levels of nitrogen were achieved by applying an 8mM inorganic nitrogen solution also in the form of KNO3, with 1mM KH2PO4, 1mM MgSO4 and microelements.

[00536] Rice tissues analyzed - All 5 rice varieties selected were grouped into 1 batch for each treatment. Two tissues [leaves and roots] growing at two different levels of nitrogen fertilization, 0.8 mM Nitrogen (nitrogen limiting conditions) or 8 mM Nitrogen (normal nitrogen conditions) were sampled and RNA was extracted, as described above. For convenience, each type of microarray expression information tissue received a set ID, as summarized in Table 12 below.
Table 12
Expression sets of rice transcriptome.

EXAMPLE 7 PRODUCTION OF BARLEY TRANSCRIPTOM AND HIGH PRODUCTIVITY CORRELATION ANALYSIS USING 44K BARLEY OLIGONUCLEOTIDE MICRO-ARRANGEMENT

[00537] In order to produce a high productivity correlation analysis, comparing the plant phenotype and the level of gene emission, the present inventors used a barley oligonucleotide microarray, produced by Agilent Technologies [http: // www ( dot) agilent chem (dot) com / Scripts / PDS (dot) asp? 1Page = 50879]. The array's oligonucleotide represents about 47,500 barley genes and transcripts. To define the correlations between the levels of RNA expression and parameters related to the components of production or vigor, several characteristics of plants from 25 different barley accessions were analyzed. Among them, 13 accessions encompassing the observed variation were selected for analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test [http: // www (dot) davidmlane (dot) com / hyperstat / A34739 (dot) htmll.

Experimental Procedures

[00538] Barley tissues analyzed - Five tissues at different stages of development [meristem, flower, spikelet, stem and leaf] that represent different characteristics of the plant were sampled and the RNA was extracted, as described above. Each type of microarray expression information tissue received a Set ID, as summarized in Table 13 below.
Table 13
Barley transcriptome expression sets

[00539] Evaluation of parameters related to the components of production and vigor of Barley - 25 accessions of barley in 4 repetitive blocks (called A, B, C and D), each containing 4 plants per lot were grown in a greenhouse with a net. The plants were phenotyped on a daily basis, following the standard barley descriptor (Table 14, below). The harvest was carried out while 50% of the ears were dried to avoid spontaneous release of the seeds. The plants were separated for the vegetative part and ears, of them, 5 ears were threshed (grains were separated from the glumes) for additional grain analysis such as size measurement, grain count by spike and grain production by spike. All the material was dried in the oven and the seeds were threshed manually from the ears before measuring the seed characteristics (weight and size) using scanning and image analysis. The image analysis system included a personal computer (Intel P43.0 GHz processor) and a public domain program - ImageJ 1.37 (Java-based image processing program, developed at the National Institute of Health in the United States and freely available on the internet [http: // rsbweb (dot) nih (dot) gov /]. Then, the analyzed data were saved as text files and processed using the JMP statistical analysis software (Instituto SAS).
Table 14
Standard barley descriptors

[00540] Grains per ear - At the end of the experiment (50% of the ears were dried), all ears of the lots within the A-D blocks were collected. The total number of grains from the 5 ears that were manually threshed was counted. The average grain per ear was calculated by dividing the total number of grain by the number of ears.

[00541] Average grain size (cm) - At the end of the experiment (50% of the ears were dried), all ears of the lots within blocks A-D were collected. The total grains of 5 ears that were manually threshed were digitized and the images were analyzed using the digital image formation system. The digitization of the grain was made using the Brother scanner (model DCP-135), at a resolution of 200 dpi and analyzed with the software Image J. The average grain size was calculated by dividing the total grain size by the total number of the grain.

[00542] Average grain weight (mg) - At the end of the experiment (50% of the ears were dried), all ears of the lots within blocks A-D were collected. The total grains of the 5 ears that were manually threshed were counted and weighed. The average weight was calculated by dividing the total weight by the total number of the grain.

[00543] Grain production per ear (g) - At the end of the experiment (50% of the ears were dried), all ears of the lots within blocks A-D were collected. The total grains of 5 ears that were manually threshed were weighed. Grain yield was calculated by dividing the total weight by the number of the ear.

[00544] Ear length analysis - At the end of the experiment (50% of the ears were dried), all ears of the lots within blocks A-D were collected. The five ears chosen per plant were measured using a tape measure excluding the edges.

[00545] Analysis of the number of ears - At the end of the experiment (50% of ears were dried), all ears of the lots within blocks A-D were collected. The ears per plant were counted.

[00546] Cultivation habit score - In cultivation stage 10 (initialization), each of the plants was scored for their cultivation habit nature. The scale that was used was 1 for prostrate nature up to 9 for upright.

[00547] Hairiness of basal leaves - In cultivation stage 5 (leaf covering strongly erect; end of tillering), each of the plants was scored for its leaf hairy nature before the last one. The scale that was used was 1 for prostrate nature up to 9 for upright.

[00548] Plant height - At the harvest stage (50% of the ears were dried), each plant was measured by its height using a tape measure. The height was measured from the ground level to the top of the longest ear, excluding the edges.

[00549] Flowering days -Each of the plants was monitored for flowering date. Flowering days were calculated from the date of sowing to the date of flowering.

[00550] Stem pigmentation - In cultivation stage 10 (initialization), each plant was scored for its stem color. The scale that was used was 1 for green to 5 for total purple.

[00551] Dry vegetable weight and ear production - At the end of the experiment (50% of the ears were dried), all ears and vegetative material from the lots within the A-D blocks were collected. The weight of the biomass and ears of each lot was separated, measured and divided by the number of plants.

[00552] Dry weight = total weight of the vegetative part above the soil (excluding roots) after drying at 70 ❑C in the oven for 48 hours;
Ear production per plant = total ear weight per plant (g) after drying at 30❑C in the oven for 48 hours.

[00553] Harvest index (for barley) - the harvest index was calculated using Formula XIII.

[00554] Formula XIII: Harvest index = average dry weight of the ear per plant / (average dry weight of the plant per plant + average dry weight of the ear per plant).
Table 15
Parameters related to Barley (vectors)

Experimental Results

[00555] 13 different accessions of barley were cultivated and characterized by 13 parameters, as described above. The mean for each of the measured parameters was calculated using the JMP software and the values were summarized in Tables 16 and 17 below. The subsequent correlation analysis between the various transcriptome expression sets (Table 13) and the mean parameters was conducted. Then, the results were integrated into the database.
Table 16
Measured parameters of correlation ID's in barley accessions

[00556] Table 16. The values of each of the parameters measured in barley accessions are provided, according to the following correlation identifications (Correlation ID's): 6 = Spikes per plant; 10 = Days for flowering; 3 = Grain weight; 5 = Ear length; 2 = Grain size; 1 = Grains per ear; 7 = Cultivation habit.
Table 17
Barley accesses, additional measured parameters (lines 713)

[00557] Table 17. The values of each of the parameters measured in barley accessions are provided, according to the following correlation identifications (Correlation ID's): 8 = Hairiness of basal leaves; 9 = Height of the plant; 4 = Grain production per ear; 11 = Pigmentation of the stem; 12 = Vegetable dry weight; 13 = Harvest index.

EXAMPLE 8 PRODUCTION OF BARLEY TRANSCRIPTOM AND HIGH PRODUCTIVITY CORRELATION ANALYSIS USING 60K BARLEY OLIGONUCLEOTIDE MICRO-ARRANGEMENT

[00558] To produce a high productivity correlation analysis, comparing the plant phenotype and the level of gene expression, the present inventors used a barley oligonucleotide microarray, produced by Agilent Technologies [http: // www (dot) chem. (dot) agilent (dot) com / Scripts / PDS (dot) asp? 1Page = 50879]. The array's oligonucleotide represents about 60,000 Barley genes and transcripts. To define the correlations between the levels of RNA expression and the parameters related to production or vigor, several plant characteristics of 15 different Barley accessions were analyzed. Among them, 10 accessions encompassing the observed variation were selected for the analysis of RNA expression. The correlation between the RNA levels and the parameters characterized was analyzed using the Pearson correlation test [Http: // www (dot) davidmlane (dot) com / hyperstat / A34739 (dot) html].

Experimental Procedures

[00559] Barley tissues analyzed - Four tissues in different stages of
development [leaf, meristem, tip of the root and adventitia root], representing different characteristics of the plant, were sampled and the RNA was extracted, as described above. Each type of microarray expression information tissue received a pool ID, as summarized in Table 18 below.
Table 18
Barley transcriptome expression sets

[00560] Table 18. Barley transcriptome expression sets are provided.

[00561] Evaluation of parameters related to the components of production and vigor of Barley - 15 accessions of Barley in 5 repetitive lots, each containing 5 plants per lot, were grown in the network greenhouse. Three different treatments were applied: the plants were fertilized and watered regularly during the growth of the plant until harvest (as recommended for commercial growth, the plants were irrigated 2 to 3 times a week, and fertilization was given in the first 1, 5 months of growth period) or with low Nitrogen (80% percent less Nitrogen) or under drought stress (drought and re-irrigation cycles were conducted throughout the experiment, total 40% less water given in dry treatment). The plants were phenotyped daily according to the parameters listed in Table 19 below. Harvesting was carried out while all ears were dried. All the material was dried in an oven and the seeds were threshed manually from the ears before measuring the characteristics of the seed (weight and size) using scanning and image analysis. The image analysis system includes a personal computer (Intel P4 3.0 Ghz processor) and a public domain program - ImageJ 1.37 (Java based on the image processing program, which was developed at the National Institutes of Health in the United States and freely available on the internet [Hypertext Transfer Protocol: // rsbweb (dot) nih (dot) gov /]. Afterwards, analysis data were saved to text files and processed using the statistical analysis software JMP (instituto SAS).

[00562] Grain Production (gr.) - At the end of the experiment, all ears of the lots were collected. The total grain from all ears that were threshed manually was weighed. Grain production was calculated by lot or by plant.

[00563] Analysis of the length and width of the ear - At the end of the experiment, the length and width of the five ears chosen per plant were measured using a tape measure, excluding the edges.

[00564] Analysis of the number of ears - The ears per plant were counted.

[00565] Height of the plant -Each of the plants was measured when its height, using a tape measure. The height was measured from the ground level to the furthest top of the ear, excluding the edges at two points of time in plant growth (30 days after sowing) and at harvest.

[00566] Ear weight - The weight of the biomass and ears of each unit was separated, measured and divided by the number of plants.

[00567] Dry weight = total weight of the plant part above the ground (excluding the roots) after drying at 70❑0 in the greenhouse for 48 hours at two points of time in Vegetable growth (30 days after sowing) and at harvest.

[00568] Spike per ear = number of spikes per ear has been counted.

[00569] Root / Sprout Ratio - The Root / Sprout ratio is calculated using Formula XIV.
Formula XIV:
Root / Sprout Ratio = total root weight at harvest / total weight of the plant part above ground at harvest.
Total No. of Profiles - all tillers were counted per unit at two time points in plant growth (30 days after sowing) and at harvest.

[00570] Percentage of reproductive tillers - the number of reproductive tillers excluding an ear at harvest was divided by the total number of tillers.

[00571] SPAD - The chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and the measurement was performed at the time of flowering. The SPAD measurement readings were taken on a new, fully developed sheet. Three measurements per sheet were taken per batch.

[00572] Root FW (gr.), Root length (cm) and N ° of lateral roots - 3 plants per unit were selected to measure root weight, root length and to count the number of lateral roots formed.

[00573] FW do Broto (fresh weight) - weight of 3 plants per pot was recorded at different time points.

[00574] Average Grain Area (cm2) - At the end of the growing period, the grains were separated from the ear. A sample of -200 grains was weighed, photographed and the images were processed using the image processing system described below. The grain area was measured from those images and was divided by the number of grains.

[00575] Average Grain Length and Width (cm) - At the end of the growing period, the grains were separated from the ear. A sample of -200 grains was weighed, photographed and the images were processed using the image processing system described below. The sum of the length and width of the grains (longest axis) was measured from those images and was divided by the number of grains.

[00576] Average Grain Perimeter (cm) - At the end of the growth period, the grains were separated from the ear. A sample of -200 grains was weighed, photographed and the images were processed using the image processing system described below. The sum of the grain perimeter was measured from those images and was divided by the number of grains.

[00577] Description date - the day on which the initialization stage was observed was recorded and the number of days from sowing to description was calculated.

[00578] Relative water content - the fresh weight (FW 1 fresh weight) of three leaves of three plants from each of the different seed ID's was recorded immediately; then, the leaves were soaked for 8 hours in distilled water at room temperature in the dark and the turgid weight (TWlturgid weight) was recorded. The total dry weight (DWIdry weight) was recorded after drying the leaves at 60 ° C at a constant weight. The relative water content (RWCIrelative water content) was calculated according to Formula I above.

[00579] Harvest Index (for barley) - The harvest index is calculated using Formula XIII above.

[00580] Relative growth rate: the relative growth rates (RGR 1 relative growth rate) of Plant Height (Formula XV below), SPAD (Formula XVI) and number of tillers (Formula XVII) were calculated as follows:
Formula XV:
Relative growth rate of plant height = regression coefficient of plant height over time.
Formula XVI:
Relative growth rate of SPAD = regression coefficient of SPAD measures over time.
Formula XVII:
Relative growth rate of Number of tillers - regression coefficient of Number of tillers over time.

[00581] Dry / Normal Ratio: Represents the proportion for the specific parameter of the Dry condition results divided by the results of the Normal conditions (maintenance of dry phenotype compared to normal conditions).
Table 19
Parameters related to Barley (vectors)

[00582] Table 19. The parameters related to Barley are provided. "TP [time point]" = time point; "DW" = dry weight; "FW" = fresh weight; "Low N" = Low Nitrogen; "Relative water content [percent] Drought / Normal Ratio" - maintenance of phenotype under drought conditions compared to normal conditions.

Experimental results

[00583] 15 different accessions of Barley were cultivated and characterized by different parameters, as described above. Table 19 describes the parameters related to Barley. The average for each of the measured parameters was calculated using the JMP software and the values are summarized in Tables 20 to 29 below. The subsequent correlation analysis between the various transcriptome sets and the average parameters was conducted. Then, the results were integrated into the database.
Table 20
Measured parameters of correlation IDs in Barley accessions under drought conditions

[00584] Table 20. The values of each of the parameters (as described above) measured in the accessions of Barley (row) of cultivation under drought conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 21
Measured parameters of correlation IDs in additional Barley accesses under drought conditions

[00585] Table 21. The values of each of the parameters (as described above) measured in the accessions of Barley (line) of cultivation under drought conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 22
Additional parameters measured from correlation IDs in Barley accessions under drought conditions

[00586] Table 22. The values of each of the parameters (as described above) measured in the accessions of Barley (row) of cultivation under drought conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 23
Additional parameters measured from the correlation IDs in the additional accesses of Barley under drought conditions

[00587] Table 23. The values of each of the parameters (as described above) measured in the accessions of Barley (row) of cultivation under drought conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 24
Measured parameters of correlation IDs in Barley accessions under normal conditions

[00588] Table 24. The values of each of the parameters (as described above) measured in the Barley accessions (line) under normal cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 25
Measured parameters of the correlation IDs in the additional accesses of Barley under normal conditions

[00589] Table 25. The values of each of the parameters (as described above) measured in the Barley accessions (line) under normal cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 26
Additional parameters measured from correlation IDs in Barley accessions under normal conditions

[00590] Table 26. The values of each of the parameters (as described above) measured in the Barley accessions (line) under normal cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 27
Measured parameters of the correlation IDs in the additional accesses of Barley under conditions of Low N.

[00591] Table 27. The values of each of the parameters (as described above) measured in the Barley accessions (line) under low nitrogen cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 28
Measured parameters of the correlation IDs in the additional accesses of Barley under conditions of Low N.

[00592] Table 28. The values of each of the parameters (as described above) measured in the Barley accessions (line) under low nitrogen cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 29
Additional parameters measured from the correlation IDs in Barley accessions under Low N. conditions

[00593] Table 29. The values of each of the parameters (as described above) measured in the Barley accessions (line) under low nitrogen cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.

EXAMPLE 9 PRODUCTION OF SORGHUM TRANSCRIPTOM AND HIGH PRODUCTIVITY CORRELATION ANALYSIS WITH PARAMETERS RELATED TO PRODUCTION, NUE AND ABST MEASURED IN FIELDS USING 44K SORGHUM OLIGONUCLEOTIDE MICRO-ARRANGEMENTS

[00594] In order to produce a high productivity correlation analysis between the plant phenotype and the level of gene expression, the present inventors used a sorghum oligonucleotide microarray, produced by Agilent Technologies [http: // www (dot ) agilent chem (dot) com / Scripts / PDS (dot) asp? 1Page = 50879]. The array's oligonucleotide represents about 44,000 sorghum genes and transcripts. In order to define the correlations between the levels of RNA expression and parameters related to vigor or production components, ABST and NUE, several plant characteristics of 17 different sorghum hybrids were analyzed. Among them, 10 hybrids encompassing the observed variation were selected for analysis of RNA expression. The correlation between the RNA levels and the characterized parameters was analyzed using Pearson's correlation test [http: // www (dot) davidmlane (dot) com / hyperstat / A34739 (dot) html].

[00595] Correlation of sorghum varieties through ecotypes grown under regular growing conditions, severe drought conditions and low nitrogen conditions.

Experimental Procedures

• 1. Condições regulares de cultivo: as plantas de sorgo foram cultivadas no campo utilizando fertilização comercial e protocolos de irrigação (370 litros por m2, fertilização de 14 unidades de 21% de ureia por período de cultivo inteiro).

[00596] 17 sorghum varieties were grown in 3 repetitive lots, in the field. Briefly, the cultivation protocol was as follows:

• 1. Regular cultivation conditions: the sorghum plants were grown in the field using commercial fertilization and irrigation protocols (370 liters per m2, fertilization of 14 units of 21% urea per entire cultivation period).

[00597] 2. Dry conditions: sorghum seeds were sown in the soil and grown in normal condition until about 35 days after sowing, around stage V8 (eight green leaves are fully expanded, initialization has not started yet). At that point, irrigation was stopped, and severe drought stress was developed.

[00598] 3. Low nitrogen fertilization conditions: the sorghum plants were fertilized with 50% less nitrogen in the field than the amount of nitrogen applied in the treatment of regular cultivation. All fertilizers were applied before flowering.

[00599] Sorghum tissues analyzed - all 10 selected sorghum hybrids were sampled for each treatment. The tissues [leaf, flower meristem and flower] of plants grown under normal conditions, severe drought stress and low nitrogen conditions were sampled and RNA was extracted, as described above. Each type of microarray expression information tissue received a Set ID, as summarized in Table 30 below.
Table 30
Expression sets of sorghum transcriptome in field experiments

[00600] Table 30: Sorghum transcriptome expression sets are provided. Leaf = the leaf below the flower; Flower meristem = apical meristem following panicle initiation; Flower = the flower on the day of anthesis.

[00601] The following parameters were collected using a digital imaging system:
Average Grain Area (cm2) - At the end of the cultivation period, the grains were separated from the 'Head' of the Plant. A sample of -200 grains was weighed, photographed and the images were processed using the image processing system described below. The grain area was measured from those images and was divided by the number of grains.

[00602] Average Grain Length (cm) - At the end of the cultivation period, the grains were separated from the 'Head' of the Plant. A sample of -200 grains was weighed, photographed and the images were processed using the image processing system described below. The sum of the lengths of the grains (longest axis) was measured from those images and was divided by the number of grains.

[00603] Average Head Area (cm2) - At the end of the cultivation period, 5 'Heads' were photographed and the images were processed using the image processing system described below. The 'head' area was measured from those images and was divided by the number of 'heads'.

[00604] Average Head Length (cm) - At the end of the cultivation period, 5 'Heads' were photographed and the images were processed using the image processing system described below. The length of the 'Head' (longest axis) was measured from those images and was divided by the number of 'heads'.

[00605] An image processing system was used, consisting of a personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.37, Java-based image processing software, developed at the National Institutes of Health from the United States and freely available on the internet at http: // rsbweb (dot) nih (dot) gov /. The images were captured in a resolution of 10 Mega Pixels (3888x2592 pixels) and stored in a low compression JPEG (Joint Photographic Experts Group standard 1 standard of the Joint Group of Specialists in Photography). Then, the image processing output data for the seed area and seed length were saved to text files and analyzed using the JMP statistical analysis software (Instituto SAS).

[00606] Additional parameters were collected either by sampling 5 plants per lot or by measuring the parameter across all plants within the lot.

[00607] Total Seed Weight per Head (g) - At the end of the experiment ('Heads' of the plant), the heads of the lots within blocks A-C were collected. 5 heads were threshed separately and the grains were weighed, all additional heads were threshed together and also weighed. The average weight of the grain per head was calculated by dividing the total weight of the grain by the total number of heads per lot (based on the lot). In the case of the 5 heads, the total weight of the 5-head grains was divided by 5.

[00608] FW head per gram of the Plant - At the end of the experiment (when the heads were harvested), the total heads and 5 heads selected by lots within blocks A-C were collected separately. The heads (total and 5) were weighed (gr.) Separately and the average fresh weight per plant was calculated for the total of heads (FW head / Gr. Plant based on the lot) and for 5 heads (FW head / Gr. Plant) based on 5 plants).

[00609] Height of the Plant - The plants were characterized with respect to the height during the period of cultivation in 5 points in time. In each measurement, the plants were measured in relation to their height using a measuring tape. The height was measured from the ground level to the top of the longest leaf.

[00610] Number of leaves of the plant - The plants were characterized in relation to the number of leaves during the period of cultivation at 5 points in time. In each measurement, the plants were measured in relation to their number of leaves by counting all the leaves of 03 plants selected per lot.

[00611] Relative Growth Rate - was calculated using Formulas XV (above) and VI (above).

[00612] SPAD - The chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and the measurement was performed 64 days after sowing. The readings of the SPAD meter were made on a new, fully developed sheet. Three measurements per sheet were made per batch.

[00613] Fresh vegetable weight and heads - At the end of the experiment (when the inflorescences were dry), all the inflorescence and plant material from the lots within blocks A-C were collected. The weight of the biomass and heads of each batch was separated, measured and divided by the number of heads.

[00614] Fresh weight = total weight of the vegetable portion above the ground (excluding the roots) after drying at 70 'C in an oven for 48 hours.

[00615] Harvest Index (HI) (Sorghum) - The harvest index was calculated using Formulas XVIII.
Formulas XVIII:
Harvest index = average dry weight of the grain per head / (average dry weight per head + average dry weight of head).

[00616] FW heads / (FW heads + FW plants) - The total fresh weight of the heads and their respective plant biomasses were measured on the day of harvest. The weight of the heads was divided by the sum of the weights of the heads and plants.

Experimental Results

[00617] 17 different sorghum hybrids were cultivated and characterized by different parameters (Table 31). The mean for each of the measured parameters was calculated using the JMP software (Tables 32-36) and a subsequent correlation analysis was conducted. The results were then integrated into the database.
Table 31
Parameters correlated to Serum () (vectors)

parâmetros (conforme descrito acima) medidos em acessos de Sorgo (ID de Semente) sob condições normais. As condições de cultivo são especificadas na seção de procedimento experimental.
Tabela 33
Parâmetros adicionais medidos em acessos de Sorgo sob condições normais de cultivo

[00618] Table 31. The parameters related to Sorghum (vectors) are provided. "g" = grams; "SPAD" = chlorophyll levels; "FW" = Fresh weight of the plant; "DW" = Dry weight of the Plant; "normal" = standard culture conditions; "DPS" = days after sowing; "Low N" = Low Nitrogen. Head - FW / Planta g. (based on 5 plants) = the fresh weight of the harvested heads was divided by the number of heads that were phenotyped; "Low N" = low nitrogen conditions; "Average Lower Rate Grain Area" = area of the grain of the lower fraction of the grains.
Table 32
Parameters measured in sorghum accesses under normal conditions

parameters (as described above) measured at Sorghum accesses (Seed ID) under normal conditions. Cultivation conditions are specified in the experimental procedure section.
Table 33
Additional parameters measured in sorghum accesses under normal growing conditions

[00619] Table 33: The values of each of the parameters (as described above) measured in Sorghum accessions (Seed ID) are given under normal conditions. Cultivation conditions are specified in the experimental procedure section.
Table 34
Parameters measured in sorghum accesses under low nitrogen conditions

[00620] Table 34: The values of each of the parameters (as described above) measured in Sorghum accessions (Seed ID) are under low nitrogen conditions. Cultivation conditions are specified in the experimental procedure section.
Table 35
Additional parameters measured in sorghum accesses under low nitrogen cultivation conditions

[00621] Table 35: The values of each of the parameters (as described above) measured in Sorghum accessions (Seed ID) under low nitrogen conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 36
Measured parameters in accesses of Sorno snh drought conditions

[00622] Table 36: The values of each of the parameters (as described above) measured in Sorghum accessions (Seed ID) under drought conditions are provided. Cultivation conditions are specified in the experimental procedure section.

EXAMPLE 10 PRODUCTION OF SORGHUM TRANSCRIPTOM AND HIGH PRODUCTIVITY CORRELATION ANALYSIS WITH PARAMETERS RELATED TO BIOMASS, NUE AND ABST MEASURED UNDER SEMI-HYDROPONIC CONDITIONS USING 44K OLIGONUCLEOTIDE MICRO ARRANGEMENTS.

[00623] Parameters related to Sorghum vigor under conditions of low nitrogen, 100 mM NaCl, low temperature (10 ± 2 ° C) and normal growing conditions - Ten sorghum hybrids were grown in 3 repetitive lots, each containing 17 plants , in a network greenhouse under semi-hydroponic conditions. In short, the cultivation protocol was as follows: Sorghum seeds were sown in trays filled with a mixture of vermiculite and peat in a 1: 1 ratio. After germination, the trays were transferred to the high salinity solution (100 rnM NaCl, in addition to the Complete Hogland solution), at low temperature (10 2 ° C, in the presence of the Complete Hogland solution), low nitrogen solution ( the amount of total nitrogen was reduced by 90% from the Complete Hogland solution (ie, to a final concentration of 10% from the Complete Hogland solution, final amount of 1.2 mM N) or in culture solution Normal [Complete Hogland's solution containing 16 mM N, at 28 ± 2 ° C]. Plants were grown at 28 ° C.

[00624] The Complete Hogland solution consists of: KNO3 - 0.808 grams / liter, MgSO4 - 0.12 grams / liter, KH2PO4 0.172 grams / liter and 0.01% (volume / volume) of the "Super coratin" microelements ( Ferro-EDDHA [ethylenediamine-N, N'-bis (2-hydroxyphenylacetic acid)] - 40.5 grams / liter; Mn - 20.2 grams / liter; Zn 10.1 grams / liter; Co 1.5 grams / liter, and Mo 1.1 grams / liter), the pH of the solution should be 6.5-6.8].

[00625] Sorghum tissues analyzed - All 10 selected Sorghum hybrids were sampled for each treatment. Three tissues [leaves, meristems and roots] grown in 100 mM NaCl, at low temperature (10 2 ° C), low nitrogen (1.2 mM N) or under Normal conditions were sampled and the RNA was extracted, as described above. Each type of microarray expression information tissue received a Set ID, as summarized in Table 37 below.
Table 37
Sorghum transcriptome expression sets under semi-hydroponic conditions

[00626] Table 37: Sorghum transcriptome expression sets are provided. Cold conditions = 10 ± 2 ° C; NaCl = 100 mM NaCl; low nitrogen 1.2 mM Nitrogen; Normal conditions = 16 mM Nitrogen.

Experimental Results

[00627] 10 different sorghum hybrids were cultivated and characterized by the following parameters: "Number of leaves" = number of leaves per plant (average of five plants), "Plant height" = plant height [cm] (average of five plants), "DW Root / Plant" - dry root weight per plant (average of five plants); "Sprout DW / Plant" - dry weight of the sprout per plant (average of five plants) (Table 38); The mean for each of the measured parameters was calculated using the JMP software and the values were summarized in Tables 39-42 below. A subsequent correlation analysis was conducted. The results were then integrated into the database.
Table 38
Parameters related to Sorghum (vectors)

[00628] Table 38: The parameters related to Sorghum are provided. Cold conditions = 10 ± 2 ° C; NaCl = 100 mM NaCl; low nitrogen = 1.2 mM Nitrogen; Normal conditions = 16 mM Nitrogen; * TP-1-2-3 refers to time periods 1, 2 and 3.
Table 39
Sorghum accesses, parameters measured under low nitrogen cultivation conditions

[00629] Table 39: The values of each of the parameters (as described above) measured in Sorghum accessions (Seed ID) under low nitrogen conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 40
Sorghum accesses, parameters measured under salinity conditions (100 mM NaCl)

[00630] Table 40: The values of each of the parameters (as described above) measured in Sorghum accessions (Seed ID) under 100 mM NaCl cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 41
Sorghum accesses, parameters measured under cold conditions

[00631] Table 41: The values of each of the parameters (as described above) measured in Sorghum accessions (Seed ID) under cultivation under cold conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 42
Sorghum accesses, parameters measured under conditions of regular cultivation

[00632] Table 42: The values of each of the parameters (as described above) measured in accessions of Sorghum (Seed ID) are provided under conditions of regular cultivation. Cultivation conditions are specified in the experimental procedure section.

EXAMPLE 11 MAIZE TRANSCRIPTOM PRODUCTION AND HIGH PRODUCTIVITY CORRELATION ANALYSIS WITH PARAMETERS RELATED TO PRODUCTION AND NUE USING 60K MAIZE OLIGONUCLEOTIDE MICRO-ARRANGEMENT

[00633] In order to produce a high productivity correlation analysis, comparing the plant's phenotype and the level of gene expression, the present inventors used a corn oligonucleotide microarray, produced by Agilent Technologies [http: // www ( point) chem. (dot) agilent (dot) com / Scripts / PDS (dot) asp? 1Page = 50879]. The array's oligonucleotide represents about 44,000 corn genes and transcripts.

[00634] Correlation of maize hybrids through ecotypes grown under regular growing conditions.

Procedures

[00635] 12 corn hybrids were grown in 3 repeated lots in the field. Corn seeds were planted and the plants were grown in the field using commercial fertilization and irrigation protocols [485 m3 of water per dunam (1000 m2), 30 units of uran 21% fertilization throughout the growing period]. To define the correlations between the levels of RNA expression with stress and the parameters related to the production or vigor components, the 12 different corn hybrids were analyzed. Among them, 10 hybrids encompassing the observed variation were selected for the analysis of RNA expression. The correlation between the RNA levels and the characterized parameters was analyzed using the Pearson correlation test [http: // www (dot) davidmlane (dot) com / hyperstat / A34739 (dot) html].

Tabela 43: São fornecidos os conjuntos de expressão de transcriptoma de milho Folha = a folha abaixo da espiga principal; Meristema de flor = meristema apical após iniciação da flor masculina; Espiga = a flor feminina no dia da antese. Grão Distal = milho desenvolvendo grão a partir da área extrema do sabugo, Grão Basal = milho desenvolvendo grãos a partir da área basal do sabugo; Entrenós = entrenós localizados acima e abaixo da espiga principal na planta. TP = período de tempo.[00636] Corn Tissues analyzed - All 10 corn hybrids selected were sampled for 3 periods of time (TP2 = V6-V8, TP5 R1-R2, TP6 = R3-R4). Four types of plant tissues [Spica, leaf indicated in Table 43 as "leaf", distal part of the grain and internode] grown under normal conditions were sampled and RNA was extracted, as described above. Each type of microarray expression information tissue received a set ID, as summarized in Table 43 below.
Table 43
Corn transcriptome expression sets

Table 43: Maize transcriptome expression sets Leaf = leaf below main ear; Flower meristem = apical meristem after initiation of the male flower; Spike = the female flower on the day of anthesis. Distal Grain = corn developing grain from the extreme area of the cob, Grain Basal = corn developing grain from the basal area of the cob; Internodes = internodes located above and below the main spike in the plant. TP = time period.

[00637] The following parameters were collected using a digital imaging system:
Grain Area (cm2) - At the end of the growth period, the grains were separated from the ear. A sample of -200 grains was weighed, photographed and the images were processed using the image processing system described below. The grain area was measured from those images and was divided by the number of grains.

[00638] Grain Length and Grain Width (cm) - At the end of the growing period, the grains were separated from the ear. A sample of -200 grains was weighed, photographed and the images were processed using the image processing system described below. The sum of the length / width of the grains (longest axis) was measured from those images and was divided by the number of grains.

[00639] Ear Area (cm2) - At the end of the growth period, 5 ears were photographed and the images were processed using the image processing system described below. The ear area was measured from those images and was divided by the number of ears.

[00640] Ear Length and Width (cm) - At the end of the growth period, 5 ears were photographed and the images were processed using the image processing system described below. The length and width of the spike (longest axis) were measured from those images and was divided by the number of spikes.

[00641] An image analysis system was used, consisting of a personal computer (Intel P4 3.0 Ghz processor) and a public domain program - ImageJ 1.37, Java-based image processing program, developed at the National Institutes of Health from the United States and freely available on the internet at http: // rsbweb (dot) nih (dot) gov /. The images were captured in a resolution of 10 Mega Pixels (3888x2592 pixels) and stored in a low compression JPEG format. Then, image processing output data for the seed area and seed length were saved to text files and analyzed using JMP statistical analysis software (SAS institute).

[00642] Additional parameters were collected either by sampling 6 plants per lot or by measuring the parameter across all plants within the lot.

[00643] Normalized Grain Weight per plant (gr.) - At the end of the experiment, all ears of the lots within the AC blocks were collected. Six ears were threshed separately and the beans were weighed, all additional ears were threshed together and also weighed. The average weight of the grain per ear was calculated by dividing the total weight of the grain by the total number of ears per lot (based on the unit). In the case of 6 ears, the total weight of the grains of 6 ears was divided by 6.

[00644] FW da Espiga (gr.) - At the end of the experiment (when the ears were harvested) total and 6 ears selected by lots within blocks A-C were collected separately. Plants with (total and 6) were weighed (gr.) Separately and the average ear per plant was calculated for total (FW of the ear per lot) and for 6 (FW of the ear per plant).

[00645] Height of the plant and height of the ear - The plants were characterized by their height at harvest. In each measurement, 6 plants were measured by their height using a tape measure. Height was measured from the ground level to the top of the plant below the corn hair. The ear height was measured from the ground level to the place where the main ear is located.

[00646] Number of leaves per plant - Plants were characterized by the number of leaves during the cultivation period 5 times in time. In each measurement, the plants were measured by their leaf number counting all the leaves of 3 selected plants per lot.

[00647] Relative Growth Rate was calculated using Formulas V-XI, XV-XVII (described above).

[00648] SPAD - The chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and the measurement was performed 64 days after sowing. The SPAD measurement readings were taken on a new, fully developed sheet. Three measurements per sheet were taken per batch. Data were taken after 46 and 54 days after sowing (DPS).

[00649] Dry weight per plant - At the end of the experiment (when the inflorescence is dry) all plant material from the lots within the A-C blocks was collected.

[00650] Dry weight = total weight of the plant part above the ground (excluding the roots) after drying at 70 ° C in a drying chamber for 48 hours.

[00651] Harvest Index (HI) (Maize) - The harvest index was calculated using Formula XIX.
Formula XIX:
Harvest Index = Average dry grain weight per Ear / (Average dry vegetable weight per Ear + Average dry weight of Ear)

[00652] Spike Filled Percentage [%] - was calculated as the percentage of the Spica area with grains outside the total spike.

[00653] Diameter of the cob [cm] - The diameter of the cob without grains was measured using a ruler.

[00654] Core Row Number per Ear - The number of rows on each ear has been counted.

Experimental Results

[00655] 12 different corn hybrids were cultivated and characterized by different parameters. The correlated parameters are described in Table 44 below. The mean for each of the measured parameters was calculated using the JMP software (Tables 45-46) and a subsequent correction analysis was performed. The results were integrated into the database.
Table 44
Corn-related parameters (vectors)

[00656] Table 44. SPAD 46DPS and SPAD 54DPS: Chlorophyll level after 46 and 54 days after sowing (DPS). "FW" = fresh weight; "DW" = dry weight.
Table 45
Parameters measured in Maize accesses under normal conditions

[00657] Table 45. The values of each of the parameters (as described above) measured in maize accessions (Seed ID) under regular cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 46
Additional parameters measured in Maize accessions under regular growing conditions

[00658] Table 46. The values of each of the parameters (as described above) measured in Maize accessions (Seed ID) under regular cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.

EXAMPLE 12 MAIZE TRANSCRIPTOM PRODUCTION AND HIGH PRODUCTIVITY CORRELATION ANALYSIS WITH PARAMETERS RELATED TO PRODUCTION AND NUE WHEN CULTIVATED UNDER REDUCED NITROGEN FERTILIZATION USING 60K CORN OLIGONUCLEOTIDE MICRO-ARRANGEMENT

[00659] In order to produce a high productivity correlation analysis between the plant's phenotype and the level of gene expression, the present inventors used a corn oligonucleotide microarray, produced by Agilent Technologies [http: // www (dot ) chem. (dot) agilent (dot) com / Scripts / PDS (dot) asp? 1Page = 50879]. The array's oligonucleotide represents about 44,000 corn genes and transcripts. Correlation of maize hybrids through ecotypes grown under low nitrogen conditions.

Experimental Procedures

[00660] 12 corn hybrids were grown in 3 repeated lots, in the field. Corn seeds were planted and the plants were grown in the field using commercial fertilization and irrigation protocols (485 m3 of water per dunam, 30 fertilization units at 21% uran for the entire growing period) or with 50% fertilization commercial to produce low N treatment. To define the correlations between the levels of RNA expression with NUE and the parameters related to the components of production or vigor, the 12 different corn hybrids were analyzed. Among them, 11 hybrids encompassing the observed variation were selected for the analysis of RNA expression. The correlation between the RNA levels and the characterized parameters was analyzed using the Pearson correlation test [Http: // www (dot) davidmlane (dot) com / hyperstat / A34739 (dot) html].

[00661] Corn Tissues analyzed - All 10 selected corn hybrids were sampled by treatment (low N and normal conditions), in three time periods (TP2 = V6-V8 (six to eight leaves are visible, growth phase rapid and core row determination initiated), TP5 = R1-R2 (silky bubble), TP6 = R3-R4 (milky mass) Four types of plant tissues [Spike, leaf indicated in Table 47 as leaf, distal part of the grain and internode] were sampled and RNA was extracted, as described above. Each type of microarray expression information tissue received a set ID, as summarized in Table 47 below.
Table 47
Corn transcriptome expression sets

[00662] Table 47: Maize transcriptome expression sets Leaf = the leaf below the main ear; Flower meristem = apical meristem after initiation of the male flower; Spike = the female flower on the day of anthesis. Distal Grain = corn developing grains from the extreme area of the cob, Grain Basal = corn developing grains from the basal area of the cob; Internodes = internodes located above and below the main spike in the plant.

[00663] The following parameters were collected either by sampling 6 plants per lot or by measuring the parameter across all plants within the lot.

[00664] Seed Production per plant (Kg.) - At the end of the experiment, all ears of the lots within blocks A-C were collected. 6 ears were threshed separately and the beans were weighed, all additional ears were threshed together and also weighed. The average weight of the grain per ear was calculated by dividing the total weight of the grain by the total number of ears per lot (based on the unit). In the case of 6 ears, the total weight of the grains of 6 ears was divided by 6.

[00665] Ear weight per lot (gr.) - At the end of the experiment (when ears were harvested) total and 6 ears selected per lot within blocks A-C were collected separately. Plants with (total and 6) were weighed (gr.) Separately and the average ear per plant was calculated for Ear weight per lot (total of 42 plants per lot).

[00666] Height of the plant and height of the ear - The plants were characterized by their height at harvest. In each measurement, 6 plants were measured by their height using a tape measure. Height was measured from the ground level to the top of the plant below the corn hair. The ear height was measured from the ground level to the place where the main ear is located.

[00667] Number of leaves per plant - Plants were characterized by the number of leaves during the cultivation period 5 times in time. In each measurement, the plants were measured by their leaf number counting all the leaves of 3 selected plants per lot.

[00668] SPAD - The chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and the measurement was performed 64 days after sowing. The SPAD measurement readings were taken on a new, fully developed sheet. Seven measurements per sheet were taken per batch. The data were taken after once a week after sowing.

[00669] Dry weight per plant - At the end of the experiment (when the inflorescence is dry) all plant material from the lots within the A-C blocks was collected.

[00670] Dry weight = total weight of the plant part above the ground (excluding the roots) after drying at 70 ° C in a drying chamber for 48 hours;
Spike Length of Filled Spike [cm] - was calculated as the length of the spike with grains outside the total spike.

[00671] Spike length and width [cm] - was calculated as the spike length and width in the filled. Measurement was performed on 6 plants per lot.

[00672] Core Row Number per Ear - The number of rows on each ear has been counted.

[00673] Stem width [cm] - The stem diameter was measured in the internode located below the main ear. Measurement was performed on 6 plants per lot.

[00674] Leaf area index [LAI I leaf area index] = total leaf area of all plants in a lot. The measurement was performed using a leaf area meter.

[00675] NUE [kg / kg] - is the proportion between the total grain production per total N applied to the soil.

[00676] NUpE [kg / kg] - is the ratio between the total plant biomass per total N applied to the soil.

[00677] Production / stem width [kg / cm] - is the ratio between total grain yields and the width of the stem.

[00678] Production / LAI [kg] - is the ratio between the total grain yields and the index of the total leaf area.

Experimental Results

[00679] 11 different corn hybrids were cultivated and characterized by different parameters. Table 48 describes the parameters related to Maize. The mean for each of the measured parameters was calculated using the JMP software (Tables 49-50) and a subsequent correction analysis was performed. The results were integrated into the database.
Table 48
Corn-related parameters (vectors)

[00680] Table 48. "cm" = centimeters "mm" = millimeters; "kg" = kilograms; SPAD in R1-R2 and SPAD R3-R4: Chlorophyll level after early and late stages of grain filling; "NUE" = efficiency in the use of nitrogen; "NUpE" = efficiency in the intake of nitrogen; "LAI" = leaf area; "N" = nitrogen; Low N = under low nitrogen conditions; "Normal" - under normal conditions; "dunam" = 1000 m2.
Table 49
Parameters measured in Maize accessions under normal fertilization

[00681] Table 49. The values of each of the parameters (as described above) measured in Maize accessions (Seed ID) under low nitrogen fertilization are provided. Cultivation conditions are specified in the experimental procedure section.
Table 50
Parameters measured in Maize accessions under low nitrogen fertilization

[00682] Table 50: The values of each of the parameters (as described above) measured in Maize accessions (Seed ID) under low nitrogen fertilization are provided. Cultivation conditions are specified in the experimental procedure section.

EXAMPLE 13 PRODUCTION OF 60K CORN TRANSCRIPTOMA OLIGONUCLEOTIDE MICRO-ARRANGEMENTS

[00683] Genes under differential display associated with Efficiency in the Use of Agronomic Nitrogen. Two commercial maize hybrids and 2 pure maize rows were grown in 5 repetitive lots in the field under six different N fertilization regimes. Corn seeds were planted and the plants were grown in the field using commercial fertilization and irrigation protocols (485 m3 dunam irrigation, 30 units of 21% uran (N fertilization) for the entire growing period - normal conditions of 100 % Nitrogen). In addition, the rest of the 5 Nitrogen treatments included: 140% Normal, 50%, 30%, 10% and 0%. In order to define the association between RNA expression levels with the parameters related to production components, biomass and NUE, several indices including Agronomic NUE, 2 maize hybrids and 1 pure maize line were selected for analysis of RNA expression . The genes regulated downwards or upwards in a given N fertilization with higher parameters of Agronomic NUE or production or biomass were considered to be associated with Agronomic NUE, NUE and production.

[00684] Corn Tissues analyzed - In total, 3 corn were sampled in V12 development stage (hair) and R3 development stage (milky). The plant tissues [leaves, lower and upper internodes, flower] were sampled and the RNA was extracted, as described above. Each type of microarray expression information tissue received a set ID, as summarized in Table 51 below.
Table 51
Corn transcriptiotomy expansion sets

[00685] Table 51: Maize transcriptome expression sets are provided

[00686] The following parameters were collected either by sampling 6 plants per lot or by measuring the parameter across all plants within the lot.

[00687] Grain weight (production) per plant (Kg.) - At the end of the experiment, all ears of the lots were collected. All ears of the batch were separately threshed and the grains were weighed, all additional ears were threshed together and also weighed. The average weight of the grain per ear was calculated by dividing the total weight of the grain by the total number of ears per lot (based on the unit).

[00688] Agronomic NUE (Efficiency in the Use of Agronomic Nitrogen) - The Agronomic NUE coefficient measures the plant's ability to efficiently use each additional unit of Nitrogen added as a fertilizer and was calculated using the following formula:
Formula XX:
Agronomic NUE = Production per plant (Kg.) X Nitrogen Fertilization O% Nitrogen Fertilization Production per plant (Kg.) Fertilizer

[00689] Ear weight per plant (gr.) - At the end of the experiment (when the ears were harvested) total and 6 ears selected by lots within the blocks were collected separately. The ears collected (total or e 6) were weighed (gr.) Separately and divided by the number of plants in order to obtain the average weight of the ear per plant.

[00690] Average Dry Weight - At the end of the experiment (when the inflorescence is dry) all plant material from the lots was collected and the 6 plants from each lot were weighed.

[00691] Cob width [cm] - The width of the cob without grains was measured using a caliper.

[00692] Total dry matter = total weight of the plant part above the ground (including ears, excluding roots).

[00693] Spike Filled / Integral - it was calculated as the length of the spike with grains outside the length of the total spike.

[00694] Spike Row Number - The number of rows on each spike has been counted.

[00695] Stem width [cm] - The diameter of the stem was measured in the internode located below the main ear. Measurement was performed on 6 plants per lot.

[00696] Leaf area index [LAI] = total leaf area of all plants in a lot. The measurement was performed using a leaf area meter.

[00697] Weight of 1000 grains - At the end of the experiment, all seeds from all lots were collected and measured and the weight of 1000 was calculated.

[00698] Grain Area (cm2) -At the end of the growing period, the grains were separated from the ear. A sample of -200 grains was weighed, photographed and the images were processed using the image processing system described below. The grain area was measured from those images and was divided by the number of grains.

[00699] Grain Length and Grain Width (mm) - At the end of the growing period, the grains were separated from the ear. A sample of -200 grains was weighed, photographed and the images were processed using the image processing system described below. The sum of the length / width of the grains (longest axis) was measured from those images and was divided by the number of grains.

[00700] Grain Perimeter (cm) - At the end of the growth period, the grains were separated from the ear. A sample of -200 grains was weighed, photographed and the images were processed using the image processing system described below. The sum of the grain perimeter was measured from those images and was divided by the number of grains.

[00701] Ear Area (cm2) -At the end of the growth period, 6 ears were photographed and the images were processed using the image processing system described below. The ear area was measured from those images and was divided by the number of ears.

[00702] Grain Filled Grain Area (cm2) - At the end of the growing period, 6 ears were photographed and the images were processed using the image processing system described below. The area of the ear filled grain was the ear area with grains outside the total ear area and was measured from those images and was divided by the number of ears.

[00703] Ear length, width and perimeter (cm) - At the end of the growth period, 6 ears were photographed and the images were processed using the image processing system described below. The length, width and perimeter of the ear were measured from those images and was divided by the number of ears.

[00704] An image processing system was used, consisting of a personal computer (Intel P4 3.0 Ghz processor) and a public domain program - ImageJ 1.37, Java-based image processing program, developed at the National Institutes of Health from the United States and freely available on the internet at http: // rsbweb (dot) nih (dot) gov /. The images were captured in a resolution of 10 Mega Pixels (3888x2592 pixels) and stored in a low compression JPEG format. Then, image processing output data for the seed area and seed length were saved to text files and analyzed using JMP statistical analysis software (SAS institute).

EXAMPLE 14 PRODUCTION OF TOMATO TRANSCRIPTOM AND HIGH PRODUCTIVITY CORRELATION ANALYSIS USING TOMATO 44K OLIGONUCLEOTIDE MICRO-ARRANGEMENT

[00705] In order to produce a high productivity correlation analysis between NUE-related phenotypes and gene expression, the present inventors used a tomato oligonucleotide microarray, produced by Agilent Technologies [http: // www (dot) chem ( dot) agilent (dot) com / Scripts / PDS (dot) asp? 1Page = 50879]. The array's oligonucleotide represents about 44,000 tomato genes and transcripts. In order to define the correlations between the levels of RNA expression and parameters related to vigor or production components, ABST and NUE, several plant characteristics of 18 different tomato varieties were analyzed. Among them, 10 varieties encompassing the observed variation were selected for analysis of RNA expression. The correlation between RNA levels and the characterized parameters was analyzed using Pearson's correlation test [Http: // www (dot) davidmlane (dot) com / hyperstat / A34739 (dot) html].

[00706] Correlation of tomato varieties through ecotypes grown under conditions with low Nitrogen, drought and regular cultivation

Experimental Procedures:

• 1. Condições de cultivo regular: variedades de tomate foram cultivadas sob condições normais (4-6 litros/m2 de água por dia e fertilizadas com NPK como recomendado nos protocolos para produção comercial de tomate).

[00707] 10 varieties of tomatoes were grown in 3 repetitive blocks, each containing 6 plants per lot was grown in a greenhouse with a net. Briefly, the cultivation protocol was as follows:

• 1. Regular cultivation conditions: tomato varieties were grown under normal conditions (4-6 liters / m2 of water per day and fertilized with NPK as recommended in the protocols for commercial tomato production).

[00708] 2. Low nitrogen fertilization conditions: tomato varieties were grown under normal conditions (4-6 liters / m2 per day and fertilized with NPK as recommended in the protocols for commercial tomato production) until the flowering stage. At that time, nitrogen fertilization was stopped.

[00709] 3. Drought stress: a variety of tomatoes was grown under normal conditions (4-6 liters / m2 per day) until the flowering stage. At that time, irrigation was reduced to 50% compared to normal conditions. The plants were phenotyped on a daily basis following the standard tomato descriptor (Table 53). The harvest was carried out while 50% of the fruits were red (ripe). The plants were separated for the vegetative part and fruits, of them, 2 nodes were analyzed for additional inflorescence parameters such as size, number of flowers, and inflorescence clump. The fresh weight of all vegetative material was measured. The fruits were separated by color (red vs. green) and according to the size of the fruit (small, medium and large). Then, the analyzed data were saved in text files and processed using the statistical analysis software JMP (instituto SAS). The data parameters collected are summarized in Table 53 below.

[00710] Tomato Tissues analyzed - Two tissues in different stages of development [flower and leaf], representing different characteristics of the plant, were sampled and the RNA was extracted, as described above. For convenience, each tissue type of the microarray expression information received a Set ID, as summarized in Table 52 below.
Table 52
Tomato transcriptome expression sets

[00711] Table 52: The identification numbers (ID) of each of the tomato expression sets are provided.

[00712] Table 53 provides the correlated parameters of the tomato (Vectors). The average for each of the measured parameters was calculated using the JMP software and the values were summarized in Tables 5459 below. A subsequent correlation analysis was conducted. The results were integrated into the database.
Table 53
Parameters correlated to Tomato (vectors)

[00713] Table 53. The parameters related to the tomato are provided. "gr." = grams; "FW" = fresh weight; "NUE" = efficiency in the use of nitrogen; "RWC" = relative water content; "NUpE" = efficiency in the intake of nitrogen; "SPAD" = chlorophyll levels; "HI" = harvest index (vegetable weight divided by production); "SLA" = specific leaf area (leaf area divided by the dry weight of the leaf).

[00714] Fruit Production (grams) - At the end of the experiment [when 50% of the fruits were ripe (red)], all the fruits of the lots inside the A-C blocks were collected. The total fruits were counted and weighed. The average weight of the fruits was calculated by dividing the total weight of the fruit by the number of fruits.

[00715] Production / SLA and Production / total leaf area - The fruit production divided by the specific leaf area or the total leaf area provided the measure of a productive balance and plant process.

[00716] Fresh weight of the plant (grams) - At the end of the experiment [when 50% of the fruits were ripe (red)], all the plants in the lots within the A-C blocks were collected. Fresh weight was measured (grams).

[00717] Inflorescence weight (grams) - At the end of the experiment [when 50% of the fruits were ripe (red)], two inflorescences of the lots within the A-C blocks were collected. Inflorescence weight (g) and number of flowers per inflorescence were counted.

[00718] SPAD - The chlorophyll content was determined using a Minolta SPAD 502 chlorophyll meter and the measurement was performed at the time of flowering. The readings of the SPAD meter were made on a new, fully developed sheet. Three measurements per sheet were per batch.

[00719] Water use efficiency (WUE) - can be terminated as the biomass produced by the unit's transpiration. To analyze the WUE, the water content relative to the leaf was measured in control and transgenic plants. The fresh weight (FW) was immediately recorded; then the leaves were soaked for 8 hours in distilled water at room temperature in the dark, and the swollen weight (TW) was recorded. The total dry weight (DW) was recorded after drying the leaves at 60 ❑C at a constant weight. The relative water content (RWC) was calculated according to Formula I below [(FW - DW / TW - DW) x 100], as described above.

[00720] Plants that maintained the relative high water content (RWC) compared to the control lines were considered more tolerant to drought than those that exhibited reduced relative water content.
Experimental Results
Table 54
Parameters measured in Tomato accesses under drought conditions

[00721] Table 54: The values of each of the parameters (as described above) measured in accessions of Sorghum (Seed ID) of cultivation under drought conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 55
Additional parameters measured in Tomato accesses under drought conditions

[00722] Table 55: The values of each of the parameters (as described above) measured in Tomato accessions (Seed ID) under Drought conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 56
Parameters measured in tomato accessions under low nitrogen conditions

[00723] Table 56: The values of each of the parameters (as described above) measured in Tomato accessions (Seed ID) under low nitrogen cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 57
Additional parameters measured in tomato accessions under low nitrogen conditions

[00724] Table 57: The values of each of the parameters (as described above) measured in Tomato accessions (Seed ID) under low nitrogen cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 58
Parameters measured in Tomato accesses under normal conditions

[00725] Table 58: The values of each of the parameters (as described above) measured in Tomato accessions (Seed ID) under normal cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 59
Additional parameters measured in Tomato accesses under normal conditions

[00726] Table 59: The values of each of the parameters (as described above) measured in Tomato accessions (Seed ID) under normal cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.

[00727] Correlation of early vigor characteristics through the collection of Tomato ecotypes under low nitrogen, 300 mM NaCl and normal cultivation conditions - Ten tomato hybrids were grown in 3 repetitive lots, each containing 17 plants, in a greenhouse network under semi-hydroponic conditions. In a nutshell, the cultivation protocol was as follows: The tomato seeds were sown in trays filled with a mixture of vermiculite and peat in a 1: 1 ratio. After germination, the trays were transferred to high salinity solution (300 mM NaCl in addition to the complete Hoagland solution), low nitrogen solution (the amount of total nitrogen was reduced by 90% of the complete Hoagland solution, final amount of 0.8 mM N), cold temperature solution (Hoagland Complete at 10 ° C), or Normal growth solution (Hoagland Complete containing 8 mM N solution, at 28 ± 2 ° C). The plants were grown at 28 ± 2 ° C.

[00728] Complete Hoagland's solution consists of: KNO3 - 0.808 grams / liter, MgSO4 -0.12 grams / liter, KH2PO4 - 0.172 grams / liter and 0.01% (volume / volume) of 'Super coratin' microelements ( FerroEDDHA [ethylenediamine-N, N'-bis (2-hydroxyphenylacetic acid)] - 40.5 grams / Liter; Mn - 20.2 grams / liter; Zn 10.1 grams / liter; Co 1.5 grams / liter; and Mo 1.1 grams / liter), the pH of the solution should be 6.5 -6.8].

[00729] Tomato Tissues analyzed - All 10 selected tomato varieties were sampled for each treatment. Three tissues [leaves, meristems and flowers] were sampled and the RNA was extracted, as described above. For convenience, each type of microarray expression information tissue received a set ID, as summarized in Table 60 below.
Table 60
Experimental sets of Tomato transcriptome

[00730] Table 60. Tomato transcriptome experimental sets are provided

[00731] Parameters related to tomato vigor - after 5 weeks of cultivation, the plants were harvested and analyzed by leaf number, plant height, chlorophyll levels (SPAD units, different levels of efficiency in the use of nitrogen (NUE) and biomass of the plant, then analysis data was saved to text files and processed using the statistical analysis software JMP (instituto SAS). The data parameters collected are summarized in Table 61, below.
Table 61
Parameters correlated to Tomato (vectors)

[00732] Table 61. The parameters related to the tomato are provided. "NUE" = efficiency in the use of nitrogen; "DW" = dry weight; "cm" = centimeter.

Experimental results

[00733] 10 different varieties of Tomato were grown and characterized by different parameters as described above. The average for each of the measured parameters was calculated using the JMP software and the values are summarized in Tables 62-64 below. The subsequent correlation analysis was performed. Then, the results were integrated into the database.
Table 62
Parameters measured in tomato accessions under low nitrogen conditions

[00734] Table 62. The values of each of the parameters (as described above) measured in tomato accessions (row) under low nitrogen cultivation conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 63
Parameters measured in Tomato accesses under normal conditions

[00735] Table 63. The values of each of the parameters (as described above) measured in tomato accessions (row) under normal growing conditions are provided. Cultivation conditions are specified in the experimental procedure section.
Table 64
Parameters measured in tomato accessions under salinity conditions

[00736] Table 64. The values of each of the parameters (as described above) measured in tomato accessions (line) of cultivation under salinity conditions are provided. Cultivation conditions are specified in the experimental procedure section.

EXAMPLE 15 GENE CLONING AND BINARY VECTOR GENERATION FOR PLANT EXPRESSION

[00737] To validate their role in increasing production, the selected genes were overexpressed in plants, as follows.

Cloning Strategy

[00738] The genes selected from those shown in Examples 1-14 above have been cloned into binary vectors for the generation of transgenic plants. For cloning, full length open reading frames (ORF's ▶ open reading frame) were identified. The EST groups and, in some cases, the mRNA sequences, were analyzed to identify the total open reading frame by comparing the results of several translation algorithms for proteins known from other plant species.

[00739] In order to clone the full length cDNA's, a reverse transcription (RT 1 reverse transcription) followed by the polymerase chain reaction (PCR; RT-PCR) was performed on the total RNA extracted from the leaves, roots or other tissues of the plant , grown under stress or normal / limiting conditions. Total RNA extraction, cDNA production and PCR amplification were performed using standard protocols described elsewhere (Sambrook J., EF Fritsch and T. Maniatis. 1989. Molecular Cloning. A Laboratory Manual, 2 'Ed. Frio Spring Harbor Laboratory Press, New York.) Which are well known to those skilled in the art. PCR products were purified using a PCR purification kit (Qiagen).

[00740] Generally, 2 sets of primers were prepared for the amplification of each gene, via nested PCR (if necessary). Both sets of primers were used for amplification in the cDNA. In the event that no product is obtained, a nested PCR reaction was performed. The nested PCR was performed by amplifying the gene using external primers and then using the PCR product produced as a model for a second PCR reaction, where the internal set of primers was used. Alternatively, one or two of the internal primers (s) were used to amplify 25 genes, both in the first and second PCR reactions (meaning that only 2-3 primers were assigned to a gene). To facilitate additional cloning of cDNA's, an extension of 8-12 base pairs (bplbase pairs) was added to the 5 'of each internal primer. The primer extension includes an endonuclease restriction site. The restriction sites were selected using two parameters: (a) the restriction site does not exist in the cDNA sequence; and (b) the restriction sites in the forward and reverse primers were designed so that the digested cDNA is inserted in the direction of the direction in the binary vector used for transformation.

[00741] PCR products were digested with restriction endonucleases (New England BioLabs Inc.), according to the designated locations on the primers. Each digested PCR product was inserted into a high copy vector pUC19 (New England BioLabs Inc.), or plasmids originating from that vector. In some cases, the undigested PCR product was inserted into the pCR-Blunt II-TOPO (Invitrogen) or directly into the binary vector. The digested products and the linearized plasmid vector were ligated using a T4 DNA ligase enzyme (Roche, Switzerland).

[00742] The sequencing of the inserted genes was performed using the ABI 377 sequencer (Applied Biosystems). In some cases, after confirming the sequences of the cloned genes, the cloned cDNA was introduced into a modified pGI binary vector containing the At6669 promoter (eg, pQFNc) and the NOS terminator (SEQ ID No.: 6066) via digestion with appropriate restriction endonucleases.

[00743] Several DNA sequences of the selected genes were synthesized via GeneArt [http: // www (dot) geneart (dot) com /]. The synthetic DNA was designed on silica. Suitable restriction enzyme sites have been added to the cloned sequences at the 5 'and 3' ends to allow further cloning into the desired binary vector.

[00744] The plasmid vector pPI was constructed by inserting a synthetic poly- (A) signal sequence originating from the basic plasmid vector pGL3 (Promega, GenBank Access No. U47295; nucleotides 4658-4811) at the HindIII restriction site of the binary vector pBI101 .3 (Clontech, GenBank Access No. U12640). PGI is similar to pPI, but the original gene in the spine is the GUSIntron gene and not the GUS.

[00745] The modified pGI vector [eg, pQFN, pQFNc, pQYN 6669, pQNaRP, pQFYN 25 or pQXNc] is a modified version of the pGI vector, in which the cassette is inverted between the left and right edges that the gene and its corresponding promoter are close to the right edge and the NPTII gene is close to the left edge.

[00746] At6669, the new Arabidopsis thaliana promoter sequence (SEQ ID No.: 3714) was inserted into the modified pGI binary vector, upstream of the cloned genes, followed by DNA ligation and extraction of the binary plasmid from the positive E. coli colonies , as described above.

[00747] The colonies were analyzed by PCR, using the primers that cover the insertion, which are designed to encompass the promoter and the introduced gene. Positive plasmids were identified, isolated and sequenced.

[00748] In cases where genomic DNA was cloned, the genes were amplified by PCR directly into the genomic DNA extracted from the leaf tissue, using the DNAeasy kit (Qiagen Cat. N ° 69104).
The selected genes cloned by the present inventors are provided in Table 65 below.
Table 65
Genes cloned in plasmids with high copy numbers or in binary vectors and primers used for cloning the genes

[00749] Table 65. "Polin." Polynucleotide; "Polip." polypeptide. For cloning each gene, at least 2 primers were used: Direct (Fwd) or Reverse (Rev). In some cases, 4 initiators were used: External Direct (EF 1 external forward), External Reverse (ER 1 external reverse), Nested Direct (NFInested forward) or Nested Reverse (NRInested reverse). The sequences of the primers used for cloning the genes are provided in the sequence listing. Some genes were synthetically produced via GeneArt (marked "GA").

EXAMPLE 16 TRANSFORMATION OF AGROBACTERIUM TUMEFACIENS CELLS WITH BINARY VECTORS HOUSING PUTATIVE GENES

[00750] The binary vectors described above were used to transform Agrobacterium cells. Two additional binary structures, having only the At6669 promoter or without an additional promoter, were used as negative controls.

[00751] Binary vectors were introduced into competent cells Agrobacterium tumefaciens GV301, or LB4404 (about 109 cells / mL), by electroporation. Electroporation was performed using a MicroPulser electroporator (Biorad), 0.2 cm of crucibles (Biorad) and EC-2 electroporation program (Biorad). The treated cells were cultured in LB liquid medium at 28 ❑C for 3 hours, then placed on LB agar supplemented with gentamicin (50 mg / L; for Agrobacterium GV301 strains) or streptomycin (300 mg / L; for Agrobacterium LB4404) and kanamycin (50 mg / L) at 28 ° C for 48 hours. The Abrobacterium colonies, which are developed in selective media, were further analyzed by PCR, using primers designed to cover the sequence inserted in the pPI plasmid. The resulting PCR products were isolated and sequenced, as described in Example 15 above, to verify that the correct polynucleotide sequences of the present application were properly introduced into Agrobacterium cells.

EXAMPLE 17 TRANSFORMATION OF ARABIDOPSIS THALIANA PLANTS WITH THE POLYNUCLEOTIDS OF THIS INVENTION PATENT APPLICATION

[00752] Arabidopsis thaliana Columbia plants (TO plants) were transformed using the Floral Immersion procedure described by Clough and Bent, 1998 (Floral dip: a simplified method for Agrobacteriummediated transformation of Arabidopsis thaliana. Plant J. 16 (6): 735 -43) and by Desfeux et al., 2000 (Female Reproductive Tissues Are the Primary Target of Agrobacterium-Mediated Transformation by the Arabidopsis Floral-Dip Methed. Plant Physiol, July 2000, Vol. 123, pp. 895-904), with minor modifications. In summary, To plants were sown in 250 ml pots, filled with a wet peat based culture mixture. The pots were covered with aluminum foil and a plastic bell jar, kept at 4 ° C for 3-4 days, then discovered and incubated in a culture chamber at 18-24 ° C in 16/8 hours of light cycles / dark. To plants were ready for transformation six days before anthesis.

[00753] Single colonies of Agrobacterium carrying the binary structures were generated, as described in Examples 15-16 above. Colonies were grown in LB medium supplemented with kanamycin (50 mg / L) and gentamycin (50 mg / L). The cultures were incubated at 28 ° C for 48 hours with vigorous agitation and then centrifuged at 4000 rpm for 5 minutes. The pellets comprising the Agrobacterium cells were resuspended in a transformation medium containing medium culture medium (2.15 g / L) Murashige-Skoog (Duchefa); 0.044gM benzylamino purine (Sigma); 112 gg / L of vitamins B5 Gambourg (Sigma); 5% sucrose and 0.2 ml / L of Silwet L77 (OSI Specialists, CT) in double distilled water with a pH of 5.7.

[00754] The transformation of plants To was carried out by inverting each plant in a suspension of Agrobacterium so that the plant tissue above the ground is submerged for 3-5 seconds. Each inoculated To plant was immediately placed in a plastic tray, then covered with a clean plastic bell jar to maintain moisture and was kept in the dark at room temperature for 18 hours to facilitate infection and transformation. The transformed (transgenic) plants were then covered and transferred to a greenhouse for recovery and maturation. The transgenic To plants were grown in the greenhouse for 3-5 weeks until the silicas were brown and dry. The seeds were harvested from the plants and kept at room temperature until sowing.

[00755] For the generation of transgenic IT plants. and T2 housing the genes, the seeds collected from transgenic To plants were sterilized on the surface by immersion in 70% ethanol for 1 minute, followed by immersion in 5% sodium hypochlorite and 0.05% triton for 5 minutes. The seeds sterilized on the surface were completely washed in sterile distilled water and then placed in the culture plates containing medium medium culture medium Murashige-Skoog (Duchefa); 2% sucrose; 0.8% of plant agar; 50 mM kanamycin and 200 mM carbenicillin (Duchefa). The culture plates were incubated at 4 ° C for 48 hours, then transferred to a culture room at 25 ° C for an additional week of incubation. The vital T1 Arabidopsis plants were transferred to fresh culture plates for another week of incubation. Following incubation, T1 plants were removed from the culture plates and culture plates and planted in the culture mixture contained in 250 ml pots. The transgenic plants were allowed to grow in a greenhouse to maturity. The seeds harvested from T1 plants were cultivated and grown to maturity as T2 plants in the same conditions as used for culture and growth of T1 plants.

EXAMPLE 18 EVALUATION OF TRANSGENIC ARABIDOSIS NUE UNDER LOW NITROGEN OR NORMAL NITROGEN CONDITIONS USING IN VITRO TESTS (FABRIC CULTURE) Test 1: plant cultivation under low and favorable nitrogen concentration levels

[00756] Sterile surface seeds were sown in basal medium [50% Murashige-Skoog (MS) medium supplemented with 0.8% plant agar as a solidifying agent] in the presence of kanamycin (used as a selection agent). After sowing, the plates were transferred for 2 to 3 days for stratification at 4 ° C and then grown at 25 ° C in daily cycles of 12 hours of light and 12 hours of darkness for 7 to 10 days. At this point in time, the seedlings randomly chosen were carefully transferred to plates containing MS medium (15 mM N) for treatment of normal nitrogen concentration and 0.75 mM of nitrogen for treatment of low nitrogen concentration. For experiments performed on T2 lines, each plate contained 5 seedlings from the same transgenic event and 3 to 4 different plates (replicates) for each event. For each polynucleotide of each invention, at least four to five independent transformation events were analyzed from each structure. For experiments performed on T1 lines, each plate contained 5 seedlings from the same transgenic event and 3 to 4 different plates (replicates) were planted. In total, for T1 lines, 20 independent events were evaluated. The plants expressing polynucleotides of the present patent application were compared to the average measurement of the control plants (empty vector or GUS reporter gene in the same promoter) used in the same experiment.

[00757] Digital image - A laboratory image acquisition system, consisting of a digital reflex camera (Canon EOS 300D) attached to a 55 mm focal length lens (Canon EF-S series), mounted on a scanning device reproduction (Kaiser RS), which includes 4 light units (4 x 150 Watts lighting lamp) and located in a dark room, was used to capture images of seedlings seen on agar plates.

[00758] The image capture process was repeated every 3 to 4 days, starting from day 1 to day 10 (see, for example, the images in Figures 3AB). An image analysis system was used, which consists of a personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.39 [Java-based image processing program, developed at the National Institutes of Health of United States and freely available on the internet at http: // rsbweb Similarly, the effect of the introduced gene on biomass accumulation, under ideal conditions, was determined by comparing the dry and fresh weight of the plants and that of the control plants (containing an empty vector or GUS reporter gene on the same promoter). From each structure created, 3 to 5 independent transformation events were examined in replicates.

[00759] Statistical analysis - In order to identify the genes that give the plant improved strength or improved architecture to the root, the results obtained from the transgenic plants were compared to those obtained from the control plants that were grown under identical cultivation conditions. To identify high-performance genes and structures, the results of the independent transformation events tested were analyzed separately. To assess the effect of a gene event on a control, the data were analyzed using the Student's t test and the p-value was calculated. The results were considered significant if p 0.1. The statistical JMP software package was used (Version 5.2.1, Instituto SAS Inc., Cary, NC, USA).

Experimental results

[00760] The genes presented in the following Tables were cloned in the regulation of a constitutive promoter (At6669). The evaluation of the transformation effect in a plant of each gene was performed by the performance test of a different number of transformation events. Some of the genes have been evaluated in more than one tissue culture assay. The results obtained in these second experiments were also significantly positive. The event with a value of p <0.1 was considered statistically significant.

[00761] The genes presented in Tables 66-69 showed a significant improvement in the plant's NUE since they produced larger plant biomass (fresh and dry weight of the plant; leaf area, root length and root cover) in T2 generation (Tables 66-67) or T1 generation (Tables 68- 69) when grown under limiting nitrogen growing conditions, compared to control plants that were grown under identical growing conditions. Plants that produce larger root biomass have a better chance of absorbing more nitrogen from the soil.
Table 66
Genes showing improved plant performance under nitrogen deficient conditions (T2 generation)

[00762] Table 66: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.
Table 67
Genes showing improved plant performance under nitrogen deficient conditions (T9 generation)

[00763] Table 67: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.
Table 68
Genes showing improved plant performance under nitrogen deficient conditions (T1 generation)

[00764] Table 68 "CONT" - Control; "Avg." - Average; "% Aum." = increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.
Table 69
Genes showing improved plant performance under nitrogen deficient conditions (T1 generation)

[00765] Table 69: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.

[00766] The genes listed in Tables 70-71 improved the relative growth rate of the plant (relative growth rate of leaf area, root coverage and root length) when grown under limiting nitrogen cultivation conditions, compared to plants of control (T1 and T2 generation) that were grown under identical growing conditions. Plants with a fast growth rate show a better plant establishment in soil under deficient nitrogen conditions. The fastest growth was observed when the growth rate of the leaf area, root length and root coverage was measured.
Table 70
Genes showing the plant's improved growth rate under nitrogen deficient conditions (aeration

[00767] Table 70: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.
Table 71
Genes showing improved plant growth rate under nitrogen deficient conditions (Ti generation)

[00768] Table 71: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.

[00769] The genes listed in Tables 72-75 improved the plant's NUE when grown at standard nitrogen concentration levels. These genes produced larger plant biomass (fresh and dry weight of the plant; leaf area, root cover and root length) when grown under standard nitrogen cultivation conditions, compared to those control plants that were grown under identical cultivation conditions under generation T2 (Tables 72-73) or generation T1 (Tables 74-75). The larger biomass of the plant in these growing conditions indicates the plant's high capacity to better metabolize the nitrogen present in the medium. Plants that produce larger root biomass have a better chance of absorbing more nitrogen from the soil.
Table 72
Genes showing improved plant performance under standard nitrogen cultivation conditions (T2 generation)

[00770] Table 72: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.
Table 73
Genes showing improved plant performance under standard nitrogen cultivation conditions (T2 generation)

[00771] Table 73: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.
Table 74
Genes showing improved plant performance under standard nitrogen cultivation conditions (T1 generation)

[00772] Table 74: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.
Table 75
Genes showing improved plant performance under standard nitrogen cultivation conditions (T1 generation)

[00773] Table 75: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.

[00774] The genes listed in Tables 76-77 improved the relative growth rate of the plant (RGR of leaf area, root length and root cover) when grown at standard nitrogen concentration levels. These genes produced plants that grew faster than control plants when grown under standard nitrogen cultivation conditions. The fastest growth was observed when the growth rate of the leaf area, root length and root coverage was measured.
Table 76
Genes showing improved plant growth rate under standard nitrogen cultivation conditions (generation T2)

[00775] Table 76: "CONT" - Control ........... significant
Table 77
Genes showing the plant's improved growth rate under standard nitrogen conditions (T1 generation)

[00776] Table 77: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.

EXAMPLE 19 EVALUATION OF NUE, PRODUCTION AND GROWTH RATE OF TRANSGENIC ARABIDOPSIS PLANT IN LOW NITROGEN OR NORMAL FERTILIZATION IN GREENHOUSE TEST

[00777] Test 1: Efficiency in the use of nitrogen: Seed production, plant biomass and plant growth rate in an optimum and limited nitrogen concentration under greenhouse conditions (greenhouse seed maturation) - This test accompanies the production of seed, biomass formation and rosette area growth of plants grown in the greenhouse under non-limiting and limiting (optimal) nitrogen growth conditions. Transgenic Arabidopsis seeds were sown on agar medium supplemented with DM medium and selection agent (kanamycin). Transgenic T2 seeds were then transplanted to 1.7 trays filled with peat and perlite in a 1: 1 ratio. . The trays were irrigated with a solution containing nitrogen limiting conditions, which were achieved by irrigating the plants with a solution containing 1.5 mM inorganic nitrogen in the form of KNO3, supplemented with 1 mM KH2PO4, 1 mM MgSO4, 3,6 mM KC1, 2 mM CaC12 and microelements, while normal nitrogen levels were achieved by applying a 6 mM inorganic nitrogen solution also in the form of KNO3, with 1 mM KH2PO4, 1 mM MgSO4, CaC12 and microelements. All plants were grown in the greenhouse until the seeds were ripe.
The seeds were harvested, extracted and weighed. The remaining plant biomass (the above-ground tissue) was also harvested, and weighed immediately or following drying in the oven at 50 ° C for 24 hours.

[00778] Each structure was validated in its T2 generation. Transgenic plants transformed with a structure formed by an empty vector carrying the At6669 promoter and the selectable marker were used as controls.

[00779] The plants were analyzed for their total size, growth rate, flowering time, seed production, weight of 1,000 seeds, dry matter and harvest index (HI - seed / dry matter production). The performance of transgenic plants was compared to control plants grown in parallel under identical growing conditions. Fake transgenic plants that express the uidA reporter gene (GUSIntron) or without any gene, in the same promoter, were used as controls.

[00780] The experiment was planned in the distribution of nested random batch. For each gene in the present application, three to five independent transformation events were analyzed from each structure.

[00781] Digital image - A laboratory image acquisition system, consisting of a digital reflex camera (Canon EOS 300D) attached with a 55 mm focal length lens (Canon EFS series), mounted on a reproduction device ( Kaiser RS), which includes 4 light units (4 x 150 Watts lamp) was used to capture images of the plant samples.

[00782] The image capture process was repeated every 2 days, starting from day 1 after the transplant until the 15th. The same camera, placed in a custom iron assembly, was used to capture the images of larger plants seen in white vats in an environmentally controlled greenhouse. The vats are square in shape and include 1.7 liter trays. During the capture process, the vats were placed below the iron assembly, while avoiding direct sunlight and casting of shadows.

[00783] An image analysis system was used, consisting of a computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.39 [Java-based image processing program, developed at the National Institutes of Health of United States and freely available on the Internet at Hypertext Transfer Protocol: // rsbweb (dot) nih (dot) gov /]. The images were captured at a resolution of 10 Mega Pixels (3888 x 2592 pixels) and stored in a low compression JPEG format (standard Joint Photographic Experts Group). Then, analysis data were saved to text files and processed using the statistical analysis software JMP (instituto SAS).

[00784] Leaf analysis - Using digital analysis, leaf data was calculated, including leaf number, rosette area, rosette diameter and leaf blade area.

[00785] Vegetative growth rate: the relative growth rate (RGR) of the leaf number [Formula VI (described above)], rosette area [Formula VII (described above)], lot coverage [Formula IX (described above) )] and harvest index [Formula IV (described above)] were calculated as follows:
Average seed weight - At the end of the experiment, all seeds were collected. The seeds were spread on a glass tray and a picture was taken. Using digital analysis, the number of seeds in each sample was calculated.

[00786] Dry weight and seed production - Around the 80th day, the plants were harvested and left to dry at 30 ❑C in a drying chamber. The biomass and seed weight of each lot were measured and divided by the number of plants in each lot. Dry weight - total weight of the plant part above the earth (excluding the roots) after drying at 30 ° C in a drying chamber; Seed production per plant = total seed weight per plant (gr.), 1000 seed weight (the weight of 1000 seeds) (gr.).

[00787] The harvest index (HI) was calculated using Formula IV, as described above.

[00788] Percentage of oil in seeds - At the end of the experiment, all seeds from each batch were collected. The seeds from 3 lots were mixed with the soil and then mounted in the extraction chamber. 210 ml of nHexane (Cat No. 080951 Biolab Ltd.) was used as a solvent. The extraction was carried out for 30 hours over medium heat at 50 ° C. Once the extraction was completed, the n-Hexane was evaporated using the evaporator at 35 ° C under vacuum conditions. The process was repeated twice. The information obtained from the Soxhlet extractor (Soxhlet, F, Die gewichtsanalytische Bestimmung des Milchfettes, Polytechnisches J, (Dingler's) 1879, 232, 461) was used to create a calibration curve for Low Resonance NMR. The oil content of all samples was determined using Low Resonance NMR (MARAN Ultra - Oxford Instrument) and its MultiQuant software package.

[00789] Silica length analysis - On the 50th day of sowing, 30 silicas of different plants in each lot were sampled in block A. The chosen silicas were green-yellow in color and were collected from the lower parts of a stem. cultivated plant. A digital photograph was taken to determine the length of the silica.

[00790] Statistical analysis -To identify the genes that give significantly improved tolerance to abiotic stresses, the results obtained from transgenic plants were compared to those obtained from control plants. To identify high-performance genes and structures, the results of the independent transformation events tested were analyzed separately. The data were analyzed by the Student's t test and the results were considered significant if the p-value was less than 0.1. The statistical JMP software package was used (Version 5.2.1, Instituto SAS Inc., Cary, NC, USA).

[00791] Tables 78-87 summarize the observed phenotypes of transgenic plants that exogenously express gene structures using seed maturation assays in the greenhouse (GH-SM) under low nitrogen (Tables 78-82) or nitrogen conditions normal (Tables 83-87). The evaluation of each gene was performed by testing the performance of different numbers of events. The event with a value of p <0.1 was considered statistically significant.
Table 78
Genes showing improved plant performance under low nitrogen cultivation conditions under regulation of the At6669 promoter

[00792] Table 78: "CONT" - Control; "Avg." - Average; "% Aum." -% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID No.: 6052).
Table 79
Genes showing improved plant performance under low nitrogen cultivation conditions under regulation of the At6669 promoter

[00793] Table 79: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID No.: 6052).
Table 80
Genes showing improved plant performance under low nitrogen cultivation conditions under regulation of the At6669 promoter

[00794] Table 80: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID No.: 6052).
Table 81
Genes showing improved plant performance under low nitrogen cultivation conditions under regulation of the At6669 promoter

[00795] Table 81: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID No.: 6052).
Table 82
Genes showing improved plant performance under low nitrogen cultivation conditions under regulation of the At6669 promoter

[00796] Table 82: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID No.: 6052).
Table 83
Genes showing improved plant performance under normal growing conditions under regulation of the At6669 promoter

[00797] Table 83: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID No.: 6052).
Table 84
Genes showing improved plant performance under normal growing conditions under regulation of the At6669 promoter

[00798] Table 84: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID No.: 6052).
Table 85
Genes showing improved plant performance under normal growing conditions under regulation of the At6669 promoter

[00799] Table 85: "CONT" - Control; "Avg." - Average; "% Aum." -% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID No.: 6052).
Table 86
Genes showing improved plant performance under normal growing conditions under regulation of the At6669 promoter

[00800] Table 86: "CONT" - Control; "Avg." - Average; "% Aum." - increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID No.: 6052).
Table 87
Genes showing improved plant performance under normal growing conditions under regulation of the At6669 promoter

[00801] Table 87: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant. The transgenes were under transcriptional regulation of the new At6669 promoter (SEQ ID No.: 6052).

EXAMPLE 20 EVALUATION OF PRODUCTION, NUE AND GROWTH RATE OF THE TRANSGENIC ARABIDOPSIS PLANT UNDER NORMAL AND LOW FERTILIZATION IN GREENHOUSE TESTS

[00802] Test 2: Nitrogen Use Efficiency measured up to the sieving stage: plant biomass and plant growth rate in ideal and limited nitrogen concentration under greenhouse conditions - This test follows the formation of the plant's biomass and the growth of the rosette area of greenhouse-grown plants under nitrogen-limiting and non-limiting cultivation conditions. The transgenic Arabidopsis seeds were sown in agar medium supplemented with; -2 of MS medium and a selection agent (kanamycin). The transgenic T2 seedlings were then transplanted to 1.7 liter trays filled with peat and pearlite in a 1: 1 ratio. The trays were irrigated with a solution containing nitrogen limiting conditions that were obtained by irrigating the plants with a solution containing 1.5 mM inorganic nitrogen in the form of KNO3, supplemented with 1 mM KH2PO4, 1 mM MgSO4, 3,6 mM KC1, 2 mM CaC12 and microelements, while normal nitrogen levels were obtained by applying a 6 mM inorganic nitrogen solution also in the form of KNO3 with 1 mM KH2PO4, 1 mM MgSO4, 2 mM CaC12 and microelements. All plants were grown in a greenhouse until sieved. The biomass of the plant (the above-ground tissue) was weighed indirectly after harvesting the rosette (fresh weight of the plant [FW]). Then, the plants were dried in an oven at 50 ❑C for 48 hours and weighed (dry weight of the plant [DW]).

[00803] Each structure was validated in its T2 generation. Transgenic plants transformed with a structure formed by an empty vector carrying the At6669 promoter and the selectable marker were used as controls.

[00804] The plants were analyzed in relation to their total size, growth rate, fresh weight and dry matter. The performance of transgenic plants was compared to control plants grown in parallel under the same conditions. Fake transgenic plants expressing the uidA reporter gene (GUSIntron) or without any gene, on the same promoter were used as a control.

[00805] The experiment was planned in the distribution of nested random batch. For each gene in the present application, three to five independent transformation events were analyzed from each structure.

[00806] Digital Image - A laboratory image acquisition system, consisting of a digital reflex camera (Canon EOS 300D) attached with a 55 mm focal length lens (Canon EFS series), mounted on a reproduction device ( Kaiser RS), which includes 4 light units (4 x 150 Watts lamps) was used to capture images of plant samples.

[00807] The image capture process was repeated every 2 days, starting on day 1 after transplantation until the 15th. The same camera, placed in a custom iron mount, was used to capture images of larger plants enclosed in vats in an environmentally controlled greenhouse. The vats were square in shape and include 1.7 liter trays. During the capture process, the vats were placed below the iron assembly, while avoiding direct sunlight and casting the shadows.

[00808] An image analysis system was used, consisting of a personal computer (Intel P4 3.0 GHz processor) and a public domain program - ImageJ 1.39 [Java-based image processing program, developed at the National Institutes of Health from the United States and freely available on the internet at http: // rsbweb (dot) nih (dot) gov /]. The images were captured in a resolution of 10 Mega Pixels (3888 x 2592 pixels) and stored in a low compression JPEG format (Joint Photographic Experts Group standard). Then, the analyzed data were saved in text files and processed using the JMP statistical analysis software (SAS institute).
Leaf Analysis - Using digital analysis, leaf data was calculated, including leaf number, rosette area, rosette diameter, and leaf blade area.

[00809] Plant growth rate: the relative growth rate (RGR) of the leaf number (Formula VI, described above), rosette area (Formula VII described above) and lot coverage (Formula IX, described below) were calculated using the formulas indicated.

[00810] Dry and fresh weight of the plant - Around the 80th day after sowing, the plants were harvested and directly weighed to determine the fresh weight of the plant (FW) and allowed to dry at 50 ❑C in a drying chamber by about 48 hours before pressing to determine the dry weight of the plant (DW).

[00811] Statistical Analysis - To identify the genes that provide a significant improvement in tolerance to abiotic stress and efficiency in the use of nitrogen, the results obtained from transgenic plants were compared to those obtained from control plants when grown under conditions identical cultivation methods. To identify high-performance genes and structures, the results of the tested independent transformation events were analyzed separately. The data were analyzed using Student's t test and the results are considered significant if the p-value is less than 0.1. The statistical JMP software package was used (Version 5.2.1, Instituto SAS Inc., Cary, NC, USA).

[00812] The genes listed in Tables 88-89 improved the plant's NUE when grown at nitrogen-limiting concentration levels. These genes produced larger plants with a larger photosynthetic area and biomass (fresh weight, dry weight, number of leaves, rosette diameter, rosette area and lot cover) when grown under nitrogen-limiting conditions (stress due to nutrient deficiency), compared to control plants grown under identical growing conditions.
Table 88
Genes showing improved plant biomass production under nitrogen-limiting cultivation conditions

[00813] Table 88: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.
Table 89
Genes showing improved plant biomass production under nitrogen-limiting cultivation conditions

[00814] Table 89: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.

[00815] The genes listed in Table 90 improved the plant's NUE when grown at limiting nitrogen concentration levels. These genes produced plants with faster development when grown under nitrogen-limiting growing conditions, compared to control plants grown under identical conditions, as measured by the growth rate of leaf number, rosette diameter and lot coverage.
Table 90
Genes showing improved rosette growth performance under nitrogen-limiting cultivation conditions

[00816] Table 90: "CONT" - Control; "Avg." - Average; "% Aum." -% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.

[00817] The genes listed in Tables 91-92 improved the plant's NUE when grown at standard nitrogen concentration levels. These genes produced larger plants with a larger photosynthetic area and improved biomass (fresh weight, dry weight, number of leaves, rosette diameter, rosette area and lot cover) when grown under standard nitrogen conditions, compared to control plants grown under identical growing conditions.
Table 91
Genes showing improved plant biomass production under standard nitrogen cultivation conditions

[00818] Table 91: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.
Table 92
Genes showing the improved biomass production of the plant under standard nitrogen cultivation conditions.

[00819] Table 92: "CONT" - Control; "- Average;" 9 6 Aum. "=% Increase;" Val-p "- p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.

[00820] The genes listed in Table 93 improved the plant's NUE when grown at standard nitrogen concentration levels. These genes produced plants with faster development when grown under nitrogen-limiting growing conditions, compared to control plants grown under identical conditions, as measured by the growth rate of leaf number, rosette diameter and lot coverage.
Table 93
Genes showing improved rosette growth performance under standard nitrogen cultivation conditions

[00821] Table 93: "CONT" - Control; "Avg." - Average; "% Aum." =% increase; "Val-p" - p-value; L means that the p-value is less than 0.01, p <0.1 was considered significant.

[00822] Although the present application for a patent has been described in conjunction with respective specific applications, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Consequently, it is intended to cover all of these alternatives, modifications and variations that fall in the spirit and broad scope of the attached claims.

[00823] All publications, patents and patent applications mentioned in that specification are incorporated in their entirety by reference in that specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated as being incorporated. here by reference. In addition, the citation or identification of any reference in the present patent application should not be interpreted as an admission that such reference is available as a prior art to the present invention patent application. As the section headings are used, they should not be interpreted as necessarily limiting.

Claims (8)

Method for increasing efficiency in the use of nitrogen, production, biomass, growth rate, vigor, oil content, and / or tolerance to the abiotic stress of a plant, characterized by the fact that it understands to overexpress within the plant a polynucleotide defined by a sequence nucleic acid defined by SEQ ID NO: 427, encoding a polypeptide defined by SEQ ID NO: 642, or a degenerate sequence thereof that encodes said polypeptide defined by SEQ ID NO: 642, thereby increasing efficiency in the use of nitrogen, production, biomass, growth rate, vigor, oil content, and / or tolerance to plant abiotic stress. Isolated nucleic acid construct characterized by the fact that it comprises an isolated polynucleotide, comprising a nucleic acid sequence as defined by SEQ ID NO: 427, encoding the polypeptide defined by SEQ ID NO: 642, or a degenerate sequence thereof that encodes the said polypeptide defined by SEQ ID NO: 642, and a promoter to direct the transcription of said nucleic acid sequence into a plant cell, wherein said promoter is not natively associated with the isolated polynucleotide Method according to claim 1, characterized in that the nucleic acid sequence is selected from the group consisting of SEQ ID NOS: 427 and 173. Nucleic acid construct according to claim 2, characterized by the fact that said polynucleotide is selected from the group consisting of SEQ ID NOS: 427 and 173. Method according to claim 1 or 3, characterized by the fact that it also comprises the cultivation of the plant that expresses said polynucleotide under abiotic stress. Method according to any one of claims 1, 3 and 5, characterized by the fact that said abiotic stress is selected from the group consisting of salinity, osmotic stress, drought, lack of water, flooding, low temperature, high temperature, toxicity by heavy metals, anaerobiosis, nutrient deficiency, excess nutrients, air pollution and UV irradiation. Method according to claim 1 or 3, characterized in that the production comprises the production of seed and the production of oil. Method according to claim 1 or 3, characterized by the fact that it also comprises the cultivation of the plant that expresses said exogenous polynucleotide under nitrogen limiting conditions.

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